Methods and compositions for stabilizing microtubules and intermediate filaments in striated muscle cells

ABSTRACT

The present invention discloses new muscle ring finger (MURF) proteins designate MURF-1, MURF-2 and MURF-3. The genes encoding these MURFs also are provided. MURFs interact with microtubules and thus play a role in cytoskeletal function, mitosis and cell growth. Thus, the uses of MURFs in diagnosis, treatment and drug screening, in particular relation to cardiomyopathies, are described.

BACKGROUND OF THE INVENTION

This application claims benefit of priority to U.S. ProvisionalApplication Serial No. 60/219,020, filed Jul. 18, 2000. The governmentmay own rights in the present invention pursuant to grant number HL07360from the National Institutes of Health.

FIELD OF THE INVENTION

The present invention relates generally to the fields of developmentalbiology and molecular biology. More particularly, it concerns a striatedmuscle RING finger protein (MURF) involved in microtubule andintermediate filament stabilization of striated muscle cells.

DESCRIPTION OF RELATED ART

The RING-finger is an unusual type of Cys-His zinc-binding motif foundin a growing number of proteins with roles in signal transduction, genetranscription, differentiation, and morphogenesis (Borden, 1998; Saurinet al., 1996). A RING-B-box-coiled-coil (RBCC) subclass of RING-fingerproteins contains an N-terminal RING-finger followed by a single ormultiple additional zinc-finger domains, termed B-boxes, and aleucine-rich coiled-coil domain (Borden, 1998). The tripartiteorganization of these domains is evolutionarily conserved, suggesting anintegrated and functional role for this overall protein structure. Itshould also be noted that the RING-finger and B-box motifs have beenidentified based on sequence homologies and are predicted to function aszinc-binding domains. However, their precise functions have not beenfully elucidated. There is evidence suggesting that the RING-finger,B-box and coiled-coil domains mediate protein-protein interactions.

Several RBCC proteins have been implicated in oncogenesis. The RBCCmember PML becomes fused to the retinoic acid receptor alpha in acutepromyelocytic leukemia (De The et al., 1991). Similarly, the RBCCproteins BRCA1, Cb1, Rfp, TIF1, and MDM2 have been demonstrated to beoncogenic when fused to other factors through chromosomal translocationevents (Saurin et al., 1996). Other RBCC proteins have been implicatedin signal transduction, organellar biogenesis, chromosomal dynamics,viral pathogenesis, transcription, and developmental patterning (Saurinet al., 1996).

Recently, a complex congenital human disease, Opitz G/BBB syndrome, wasshown to result from mutations in the RBCC protein, Mid1 (Quaderi etal., 1997). Opitz G/BBB syndrome is characterized by abnormalities ofmidline structures, including hypertelorism, clefts of lip and palate,larygotracheoesophageal defects, hypospadias, imperforate anus, anddevelopmental delay. The Mid1 gene product is widely expressed duringdevelopment and interacts with microtubules throughout the cell cycle(Cainarca et al., 1999). Overexpression of Mid1 leads to a stablepopulation of microtubules resistant to depolymerization (Schweiger etal., 1999). Interestingly, mutations of Mid1 that are linked to OpitzG/BBB syndrome severely diminish the ability of Mid1 to interact withmicrotubules, suggesting that Mid1-microtubule interaction and/ormicrotubule dynamics are involved in the processes required for normaldevelopment of the midline structures affected in Opitz G/BBB syndrome.

Many questions remain regarding the function of Mid1-type proteins andtheir interactions with microtbules. Nonetheless, it is clear that suchmolecules play an important role in development, function and pathologyof a wide variety of cell types.

SUMMARY OF THE INVENTION

Therefore, in one aspect of the invention, there is provided a DNAsegment encoding a MURF-1, MURF-2 or MURF-3 polypeptide. The MURF-1,MURF-2 or MURF-3 polypeptide may be human, mouse, dog, rabbit, rat,Drosphila, yeast or other species. In a particular embodiment, theMURF-1 polypeptide has the sequence of SEQ ID NO:2, the MURF-2polypeptide has the sequence of SEQ ID NO:4, and the MURF-3 polypeptidehas the sequence of SEQ ID NO:6. In yet more particular embodiments, theMURF-1 DNA segment has the sequence of SEQ ID NO:1, the MURF-2 DNAsegment has the sequence of SEQ ID NO:3, and the MURF-3 DNA segment hasthe sequence of SEQ ID NO:5.

The DNA segment may be positioned under the control of a promoter, forexample, a promoter not native to the MURF-1, MURF-2 or MURF-3 codingregion. The MURF-1, MURF-2 or MURF-3 coding region gene may bepositioned in reverse orientation to the promoter, thereby capable ofexpressing an antisense product. The DNA segment may further comprise apolyadenylation signal. The DNA segment may further comprise an originof replication. The DNA segment may be viral vector or a non-viralvector.

In another aspect of the invention, there is provided a host cellcomprising a DNA segment that encodes a MURF-1, MURF-2 or MURF-3polypeptide, wherein said DNA segment comprises a promoter heterologousto the MURF-1, MURF-2 or MURF-3 coding region. The host cell may furtherbe defined as a prokaryotic host cell or a eukaryotic host cell. Thehost cell may be a secretory cell.

In yet another aspect of the invention, there is provided a method ofusing a host cell comprising an expression cassette comprising apolynucleotide encoding a MURF-1, MURF-2 or MURF-3 polypeptide and apromoter active in said host cell, said promoter directing theexpression of said polypeptide, said method comprising culturing thehost cell under conditions suitable for the expression of the MURF-1,MURF-2 or MURF-3 polypeptide.

In still yet another aspect of the invention, there is provided anisolated nucleic acid segment comprising at least 15 contiguousnucleotides of SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5. The isolatednucleic acid segment may be 15, 20, 25, 30, 35, 40, 45, 50, 75 or 100nucleotides in length. The number of contiguous nucleotides may beincreased to 20, 25, 30, 35, 40, 45, 50, 75 or 100.

In still yet an additional embodiment, there is provided as an isolatednucleic acid segment of from 14 to about 888 nucleotides in length thathybridizes to the nucleic acid segment of SEQ ID NO:1, SEQ ID NO:3 SEQID NO:5, or complements thereof, under standard hybridizationconditions. The isolated nucleic acid segment may further comprise anorigin of replication. The isolated nucleic acid may be a viral vectorselected from the group consisting of retrovirus, adenovirus,herpesvirus, vaccinia virus, poxvirus, and adeno-associated virus.Further, the isolated nucleic acid may be packaged in a liposome.

In another aspect of the invention, there is provided a nucleic aciddetection kit comprising, in suitable container means, an isolatednucleic acid segment that hybridizes under high stringency conditions tothe nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO:3 or SEQ ID NO:5,or complements thereof. The may further comprise a detection reagent,for example, a detectable label that is linked to said nucleic acidsegment.

In yet another embodiment, there is provided a composition comprising apurified MURF-1, MURF-2 or MURF-3 protein or peptide that includes acontiguous amino acid sequence from SEQ ID NO:2, SEQ ID NO:4 or SEQ IDNO:6. In still yet another embodiment, there is provided a purified MURFprotein having the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4 orSEQ ID NO:6. In still a further embodiment, there is provided arecombinant MURF-1, MURF-2 or MURF-3 protein or peptide prepared byexpressing a DNA segment that encodes a MURF-1, MURF-2 or MURF-3 proteinor peptide in a recombinant host cell and purifying the expressedMURF-1, MURF-2 or MURF-3 protein or peptide away from total recombinanthost cell components.

In another embodiment, there is provided an isolated peptide of betweenabout 10 and about 50 amino acids in length, comprising a contiguousamino acid sequence from the sequence of SEQ ID NO:2, SEQ ID NO:4 or SEQID NO:6. The peptide may be about 10, 15, 20, 25, 30, 35, 40, 45, 50, 75or 100 amino acids in length. In yet another embodiment, there isprovided an antibody composition that binds to a protein or peptide thatincludes an epitope from SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6. Theantibody composition may comprise monoclonal antibodies or polyclonalantibodies. The antibodies of the composition are operatively attachedto a detectable label, the label could be selected from the groupconsisting of a fluorescent label, a chemiluminescent label, aelcetroluminescent label, a radiolabel and an enzyme. Also provided is ahybridoma cell that produces a monoclonal antibody that bindsimmunologically to MURF-1, MURF-2 or MURF-3. Also provided is animmunodetection kit comprising, in suitable container means, a firstantibody that binds to a MURF-1, MURF-2 or MURF-3 protein or peptide.

In still yet another embodiment, there is provided a method fordetecting alterations in MURF-1, MURF-2 or MURF-3 function in a cellcomprising assessing the structure or expression level of a MURF-1,MURF-2 or MURF-3 polypeptide. The method may comprise determining thestructure of a MURF-1, MURF-2 or MURF-3 gene, for example, sequencing aMURF-1, MURF-2 or MURF-3 gene, or Southern or Northern analysis of aMURF-1, MURF-2 or MURF-3 transcript or gene. Alternatively, theassessing may comprise determining the level of a MURF-1, MURF-2 orMURF-3 protein or transcript in the cell, for example, by Northernanalysis of MURF-1, MURF-2 or MURF-3 transcripts, or immunodetection ofMURF-1, MURF-2 or MURF-3 protein levels (ELISA, Western blot).

In yet a further embodiment, there is provided a method for increasingMURF-1, MURF-2 or MURF-3 activity in cell comprising administering tothe cell with an expression construct comprising a MURF-1, MURF-2 orMURF-3 coding region under the control of a promoter active in the cell.

In still yet a further embodiment, there is provided a method ofscreening a candidate substance for MURF-1, MURF-2 or MURF-3 bindingactivity comprising (i) providing a MURF-1, MURF-2 or MURF-3polypeptide; (ii) contacting the MURF-1, MURF-2 or MURF-3 polypeptidewith the candidate substance; and (iii) determining the binding of thecandidate substance to the MURF-1, MURF-2 or MURF-3 polypeptide. Theassay may be performed in a cell free system or in a cell.

In another embodiment, there is provided a method of screening acandidate substance for an effect on MURF-1, MURF-2 or MURF-3 levels ina cell comprising (i) providing a cell that expresses MURF-1, MURF-2 orMURF-3 polypeptide; (ii) contacting the cell with the candidatesubstance; and (iii) determining the effect of the candidate substanceon MURF-1, MURF-2 or MURF-3 polypeptide level.

In still another embodiment, there is provided a method of screening acandidate substance for an effect on MURF-1, MURF-2 or MURF-3 expressionin a cell comprising (i) providing a cell that expresses MURF-1, MURF-2or MURF-3 polypeptide; (ii) contacting the cell with the candidatesubstance; and (iii) determining the effect of the candidate substanceon MURF-1, MURF-2 or MURF-3 mRNA levels.

In still yet another embodiment, there is provided a method of screeninga candidate sustance for an effect on MURF-1, MURF-2 or MURF-3interaction with microtubles comprising (i) providing a microtubulecomposition; (ii) contacting the microtubule composition with MURF-1,MURF-2 or MURF-3 polypeptide in the presence of the candidate substance;and (iii) assessing the interaction of MURF-1, MURF-2 or MURF-3 with themicrotubule composition in the presence of the candidate substance,wherein a change in the interaction of MURF-1, MURF-2 or MURF-3 with themicrotubule composition, as compared to the interaction in the absenceof the candidate substance, indicates that the candidate substancemodulates the interaction of MURF-1, MURF-2 or MURF-3 and microtubules.Step (iii) may comprise a cosedimentation assay.

In another embodiment, there is provided a method for screening acandidate substance for an effect on MURF-1, MURF-2 or MURF-3homodimeraization comprising (i) providing a MURF-1, MURF-2 or MURF-3polypeptide composition; (ii) contacting the composition with thecandidate substance; and (iii) determining the effect of the candidatesubstance on MURF-1, MURF-2 or MURF-3 homodimerization.

In still another embodiment, there is provided a method of screening acandidate substance for an effect on MURF-1, MURF-2 or MURF-3 directedglutamic acid modification of microtubules comprising (i) providing acell that expresses MURF-1, MURF-2 or MURF-3 polypeptide; (ii)contacting the cell with the candidate substance; and (iii) determiningthe effect of the candidate substance on glutamic acid modification ofmicrotubules.

In yet a further embodiment, there is provided a method of screening acandidate sustance for an effect on MURF-1, MURF-2 or MURF-3stabilization of microtubles comprising (i) providing a microtubulecomposition; (ii) contacting the microtubule composition with MURF-1,MURF-2 or MURF-3 polypeptide in the presence of the candidate substance;and (iii) assessing the stability of the microtubule composition in thepresence of the candidate substance, wherein a change in the stabilityof MURF-1, MURF-2 or MURF-3 with the microtubule composition, ascompared to the stability in the absence of the candidate substance,indicates that the candidate substance modulates the stabilitymicrotubules.

Also provided is a transgenic non-human mammal, cells of which comprisea MURF-1, MURF-2 or MURF-3 encoding nucleic acid segment integrated intotheir genome, wherein the MURF-1, MURF-2 or MURF-3 encoding nucleic acidis under the control of a heterologous promoter. The promoter may be atissue specific promoter, for example, a muscle specific promoter, suchas myosin light chain-2 promoter, alpha actin promoter, troponin 1promoter, Na⁻/Ca²⁺ exchanger promoter, dystrophin promoter, creatinekinase promoter, alpha7 integrin promoter, brain natriuretic peptidepromoter, and alpha B-crystallin/small heat shock protein promoter. Thetransgenic mammal may be a mouse.

In another embodiment, there is provided a method of treating cardiacfailure comprising increasing MURF-1, MURF-2 or MURF-3 activity in acardiac cell, wherein said increased MURF-1, MURF-2 or MURF-3 activitystabilizes microtubules and/or intermediate filaments. The method maycomprise increasing MURF-1, MURF-2 or MURF-3 activity by contacting saidcardiac cell with an expression cassette that comprises a polynucleotideencoding a MURF-1, MURF-2 or MURF-3 polypeptide and a promoter active insaid cardiac cell, wherein said promoter directing the expression ofsaid polypeptide. The promoter may bea cardiac specific promoter. Thecontacting may be by intravenous or intraarterial administration of avector comprising said expression cassette.

In yet a further embodiment, there is provided a method of decreasingMURF-1, MURF-2 or MURF-3 activity in a cell comprising administering tosaid cell an agent that inhibits MURF-1, MURF-2 and/or MURF-3 activity.The agent be a small molecule, an antisense molecule that hybridizes toMURF-1, MURF-2 and/or MURF-3 transcripts, a ribozyme molecule thatcleaves MURF-1, MURF-2 and/or MURF-3 transcripts. Also provided is amethod of blocking MURF-1, MURF-2 or MURF-3 expression in a cellcomprising administering to said cell an agent that inhibitstranscription or translation of MURF-1, MURF-2 and/or MURF-3.

In still a further embodiment, there is provided a method of screening acandidate sustance for an effect on MURF-1, MURF-2 or MURF-3 interactionwith intermediate filaments comprising (i) providing an intermediatefilament composition; (ii) contacting the intermediate filamentcomposition with MURF-1, MURF-2 or MURF-3 polypeptide in the presence ofthe candidate substance; and (iii) assessing the interaction of MURF-1,MURF-2 or MURF-3 with the intermediate filament composition in thepresence of the candidate substance, wherein a change in the interactionof MURF-1, MURF-2 or MURF-3 with the intermediate filament composition,as compared to the interaction in the absence of the candidatesubstance, indicates that the candidate substance modulates theinteraction of MURF-1, MURF-2 or MURF-3 and intermediate filament. Themethod may be in a cell or a cell free system. It may be performed invivo. The method may comprise a cosedimentation assay. The intermediatefilaments may be one or more of desmin, vimentin and cytokeratin.

In yet still a further embodiment, there is provided a method forscreening a candidate substance for an effect on MURFheterodimerizationcomprising (i) providing two or more of a MURF-1,MURF-2 or MURF-3 polypeptide composition; (ii) contacting thecompositions with the candidate substance; and (iii) determining theeffect of the candidate substance on the heterodimerization of two ormore of MURF-1, MURF-2 or MURF-3.

In each of the preceding screening embodiments, there also is providedsimilar methods for the production of a modulator, comprising each ofthe aforementioned screening steps, followed by the additional step ofproducing the modulator so identified.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIGS. 1A-1D—Amino acid sequence of MURF-1 and structural similarity toMidline proteins. FIG. 1A. Deduced amino acid sequence of MURF-1. TheRING-finger domain is boxed, the B-box domain is shaded, and thecoiled-coil domain is underlined. FIG. 1B. Schematic diagrams of MURF-1and Midline proteins. FIGS. 1C and 1D. Amino acid homologies between theRING-finger and B-box domains, respectively, of MURF-1 and Midlineproteins.

FIGS. 2A-2D—Muscle-specific expression of MURF-1 RNA and protein. FIG.2A. Detection of MURF-1 transcripts by in situ hybridization to mouseembryo sections. A transverse section through an E8.5 embryo showsexpression exclusively in the cardiac forming region. A transversesection through an E10.5 embryo shows expression in the heart andmyotomes. A sagittal section through an E16.5 embryo shows expressionthroughout skeletal muscle and heart: a, atria; m, myotome; hf, headfolds; nt, neural tube, v, ventricle. FIG. 2B. Northern analysis ofpolyA+ mRNA from adult mouse tissues. A 1.5 kb MURF-1 transcript isdetected only in heart and skeletal muscle. FIG. 2C. Western blotanalysis of MURF-1 protein in extracts from adult mouse tissues. The 41kD MURF-1 protein is detected only in heart and skeletal muscle.Blotting with anti-tubulin antibody confirmed equal quantities ofprotein in each lane. FIG. 1D. RT-PCR analysis of RNA from C2 cells ingrowth medium (GM) or differentiation medium (DM) for 1 or 3 days, asindicated. MURF-1 transcripts are detected at low levels inundifferentiated myoblasts in GM and are upregulated duringdifferentiation. L7 is expressed constitutively and is a control for RNAloading.

FIGS. 3A-3B—Association of MURF-1 with microtubules demonstrated bymicrotubule sedimentation assay. FIG. 3A. Microtubule cosedimentation ofendogenous MURF-1 protein from striated muscle. Microtubules fromsoluble extracts from striated muscle were induced to polymerize asdescribed in Material and Methods. MURF-1 is contained in themicrotubule pellet from striated muscle. FIG. 3B. Localization of MURF-1to microtubules of C2 myotubes detected by immunofluorescence withanti-M RF-1 antibody, as described in Materials and Methods. MURF-1 islocalized to filamentous microtubules of the cytoskeleton in C2myotubes.

FIGS. 4A-4G—Mapping of domains of MURF-1 required for microtubulelocalization by immunofluorescence. FIGS. 4A-C. Hela cells weretransfected with myc-tagged MURF and immunofluorescence performed.MURF-1 (green) forms filaments that directly colocalize withmicrotubules (red) as seen in panel C. Yellow color in C is the resultof direct overlap of MURF-1 and microtubule localization. FIG. 4D.Summary of the microtubule binding domain of MURF-1. The leucine-richcoiled-coil domain mediates microtubule interaction and the RING-fingeris required for filament formation. FIG. 4E. Deletion of the N-terminal16 amino acids alters the undular filaments to a more angular assembly.FIG. 4F. The RING-finger domain is required for filament formation. FIG.4G. Deletion of the C-terminal acidic domain does not affect microtubuleinteraction nor filament formation. Cos cells were used in FIGS. 4E and4G. Identical results were obtained in multiple cell types.

FIGS. 5A-5C—Mapping of domains of MURF-1 required for association withtubulin by cosedimentation. FIG. 5A. Schematic representation of theexperimental protocol used in the in vitro microtubule cosedimentationassay. 293T cells treated with nocodazole were transfected with variousmyc-tagged versions of MURF-1 and soluble extracts prepared. Purifiedtubulin was added and microtubules were polymerized in the presence ofGTP and EGTA. Polymerized microtubules were pelleted and the resultingsupernatant was precipitated. The pellet was subjected todepolymerization by calcium and cold treatment. Three cycles ofpolymerization-depolymerization were repeated before western analysis.FIG. 5B. Representation of MURF-1 deletions used in the analysis. FIG.5C. Western blot analysis of MURF-1 cosedimentation with microtubules.MURF-1 protein containing the leucine-rich coiled-coil domaincosediments with microtubules. An equivalent amount of MURF-1 mutantN212 is contained in the supernatant and microtubule pellet, lane 5 (*)indicating the amino acids 167-211 are required for optimal microtubuleassociation. The blot was reprobed for tubulin content to assess theextent of microtubule polymerization. Densitometric analysis revealedgreater than 90% of the tubulin input was contained in the microtubulepellet.

FIGS. 6A-6D—Mapping domains of MURF-1 required for homo-oligomerization.FIG. 6A. Various forms of N-terminal Flag-tagged MURF-1 and full-lengthC-terminal myc-tagged MURF-1 were in vitro translated in the presence of³⁵S-methionine and immunoprecipitation performed as described inMaterial and Methods. The leucine-rich coiled-coil domain mediateshomo-oligomerization of MURF-1. FIGS. 6B-D. MURF-1 binds to microtubulesin a homo-oligomeric form. Hela Cells were transfected with Flag-taggedfull-length MURF alone (FIG. 6B) or in combination with myc-taggedMURF-1 (FIGS. 6C and 6D) and immunofluorescence performed. Flag-taggedMURF-1 forms aggregates in the cytoplasm of the cell. In the presence ofmyc-tagged MURF-1, the Flag-tagged MURF-1 and myc-tagged MURF-1 arecolocalized in filamentous structures in the cell. Identical resultswere obtained using native MURF-1 lacking an epitope tag.

FIGS. 7A-7D—Mapping domains of MURF-1 required for microtubulestabilization. FIGS. 7A-7C. Expression of MURF-1 stabilizesmicrotubules. Cos cells were transfected with MURF-1 and treated with2∝M nocodazole for 2 hours and immunofluorescence performed. As seen inpanel C, only cells expressing MURF-1 contain intact microtubulesfollowing nocodazole treatment. FIG. 7D. Microtubule interaction andfilament formation are required for microtubule stabilization. Deletionof the RING-finger domain (mutant N81) abrogating filament formation orthe leucine-rich coiled-coil domain (mutant C199) preventingmicrotbubule association ablates the microtubule stabilization functionof MURF-1.

FIGS. 8A-8D—Stabilization of microtubules by MURF-1 in vivo. FIGS.8A-8C. MURF-1 expression enhances Glu-tubulin formation. Cos cells weretransfected with MURF-1 and immunostained for MURF-1 and Glu-tubulin todetect stable microtubules. In panel C only cells expressing MURF-1 havea microtubule network enriched in Glu-tubulin. FIG. 8D. MURF-1expression parallels the appearance of Glu-tubulin during skeletalmuscle formation in vitro. C2 cells were induced to differentiate forvarying lengths of time and western blot analysis performed to detectMURF-1 expression and Glu-tubulin. MURF-1 expression is observedimmediately after differentiation is induced. The formation ofGlu-tubulin mirrors the appearance of MURF-1 protein. Blotting with ananti-tubulin antibody confirmed equal quantities of protein in eachlane.

FIG. 9—Functional domain structure of MURF-1. MURF-1 is composed ofdistinct functional domains. The leucine-rich coiled-coil domainmediates homo-oligomerization and microtubule association. TheRING-finger domain is required for filament formation alongmicrotubules.

FIG. 10—Alignment of MURF1, MURF2 and MURF3 protein sequences.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present inventors describe herein novel RBCC proteins that showsignificant homology to Midline proteins in the RING-finger and B-boxregions. However, this protein is structurally distinct from knownMidline proteins, and its expression is restricted to cardiac andskeletal muscle throughout pre- and postnatal development. Inparticular, MURF1 is down-regulated in cardiomyopathy mouse models, butits overexpression in the heart also results in cardiac dysfunction anddeath. Like Mid1, MURFs bind and stabilizes microtubules againstdepolymerizing agents. In addition, MURFs associate with desmin,cytokeratin and vimentin. Thus, MURFs also appear to be generalintermediate filament binders. These properties suggest involvement inmicrotubule and intermediate filament stabilization in striated musclecells, which is important for alignment of skeletal myoblasts duringfusion, myofibrillar assembly, and contractile function. MURFs alsohetero-associate with each other, although the precise significance ofthese interactions is not yet clear.

I. MURF Peptides and Polypeptides

MURF is a designation assigned by the present inventors for Muscle RINGFinger proteins. While these molecules are distinct from earlier knownpolypeptides, they appear to be part of a group of structurally- andfunctionally-related molecules known as Midline proteins.

In addition to the entire MURF-1, MURF-2 and MURF-3 molecules, thepresent invention also relates to fragments of the polypeptides that mayor may not retain various of the functions described below. Fragments,including the N-terminus of the molecule may be generated by geneticengineering of translation stop sites within the coding region(discussed below). Alternatively, treatment of the MURFs withproteolytic enzymes, known as proteases, can produces a variety ofN-terminal, C-terminal and internal fragments. Examples of fragments mayinclude contiguous residues of SEQ ID NOS:2, 4 and 6 of 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40,45, 50, 55, 60, 65, 75, 80, 85, 90, 95, 100, 200, 300, 400 or more aminoacids in length. These fragments may be purified according to knownmethods, such as precipitation (e.g., ammonium sulfate), HPLC, ionexchange chromatography, affinity chromatography (includingimmunoaffinity chromatography) or various size separations(sedimentation, gel electrophoresis, gel filtration).

A. Structural Features of the Polypeptide

MURF-1 is 366 amino acid polypeptide. The predicted molecular weight ofthis molecule is 41 kDa, with a resulting pI of 4.82 Thus, at a minimum,this molecule may be used as a standard in assays where molecule weightand pl are being examined.

MURF-1 contains several domains that identify it as a RBCC-type ofRING-finger protein. A RING-finger of the C₃HC₄ type is located near theamino-terminus (amino acids 26-81), followed by another type ofzinc-finger termed a B-box (amino acids 126-158) (FIG. 1B). In all otherRBBC proteins, the spacing between the RING-finger and B-box is alsoabout 40 amino acids (Borden, 1998; Saurin et al., 1996). A predictedleucine-rich coiled-coil domain (amino acids 212-253) and an acidicregion (amino acids 335-366) are located in the C-terminal portion ofthe protein.

Database searches with the amino acid sequence of MURF-1 revealedhighest homology to the Opitz-G/BBB syndrome protein Mid1 and therelated factor Mid2 (FIG. 1C and FIG. 1D) with greatest homology in theRING-finger and B-box domains. Interestingly, MURF-1 does not containthe first B-box of Mid1 and Mid2 nor the butyrophilin-like domain at theC-termini of Mid2 and Mid2, suggesting functional differences betweenthe proteins.

B. Functional Aspects

Although the precise role played my MURFs in cellular physiology is notelucidated, a number of particular functions have been associated withMURF-1. First, MURF-1 binds both to itself (homo-oligomerization) and tomicrotubules. Microtubule-binding and homo-oligomerization are mediatedby the coiled-coil domain at the C-terminus. In addition, MURF-1 playsin role in the formation of microfilaments; this is reliant on theRING-finger domain of MURF-1.

MURF-1 is expressed specifically in cardiac and skeletal tissue. It isdownregulated in late stage failing heart, as the heart loses definedstructure and contractility. Overexpression of MURF in non-muscle cellsinhibits growth, presumably by binding and stabilizing microtubules, thedissociation of which is a prerequisite for mitosis.

The equilibrium of microtubules between the polymerized anddepolymerized states influences a variety of cellular processes,including morphological changes, migration, and proliferation. Themicrotubule-binding properties of MURF are similar in many respects tothose of other microtubule associated proteins, such as MAPs, Midlinefamily members, Doublecortin, Lisl, and Tau, which bind and stabilizemicrotubules (Buchner et al. 1999; Cainarca et al. 1999; Gleeson et al.,1999; Hsieh et al., 1999; Kaech et al, 1996; Kosik, 1990; Koulakoff etal., 1999; Nguyen et as, 1998; Nguyen et al, 1998; Sapir et al., 1997;Sapir et al., 1999; Schweiger et al, 1999; Takemura et al, 1992).

However, MURF lacks a recognizable microtubule association domaincommonly contained in MAPs but, instead, associates with microtubulesthrough its leucine-rich coiled-coil domain. Other microtubule bindingproteins that lack a classic microtubule association domain found inMAPs also have been identified, including Mid1, Mid2, Doublecortin andLisl. In this respect, MURF is a member of the non-classical MAPslacking a consensus microtubule association domain. With the exceptionof Mid1 and Mid2, most non-classical MAPs do not share significanthomology with MURF, suggesting multiple mechanisms for microtubuleinteraction that may regulate microtubule dynamics in different tissues.

C. Variants of MURFs

Amino acid sequence variants of the polypeptide can be substitutional,insertional or deletion variants. Deletion variants lack one or moreresidues of the native protein which are not essential for function orimmunogenic activity, and are exemplified by the variants lacking atransmembrane sequence described above. Another common type of deletionvariant is one lacking secretory signal sequences or signal sequencesdirecting a protein to bind to a particular part of a cell. Insertionalmutants typically involve the addition of material at a non-terminalpoint in the polypeptide. This may include the insertion of animmunoreactive epitope or simply a single residue. Terminal additions,called fusion proteins, are discussed below.

Substitutional variants typically contain the exchange of one amino acidfor another at one or more sites within the protein, and may be designedto modulate one or more properties of the polypeptide, such as stabilityagainst proteolytic cleavage, without the loss of other functions orproperties. Substitutions of this kind preferably are conservative, thatis, one amino acid is replaced with one of similar shape and charge.Conservative substitutions are well known in the art and include, forexample, the changes of: alanine to serine; arginine to lysine;asparagine to glutamine or histidine; aspartate to glutamate; cysteineto serine; glutamine to asparagine; glutamate to aspartate; glycine toproline; histidine to asparagine or glutamine; isoleucine to leucine orvaline; leucine to valine or isoleucine; lysine to arginine; methionineto leucine or isoleucine; phenylalanine to tyrosine, leucine ormethionine; serine to threonine; threonine to serine; tryptophan totyrosine; tyrosine to tryptophan or phenylalanine; and valine toisoleucine or leucine.

The following is a discussion based upon changing of the amino acids ofa protein to create an equivalent, or even an improved,second-generation molecule. For example, certain amino acids may besubstituted for other amino acids in a protein structure withoutappreciable loss of interactive binding capacity with structures suchas, for example, antigen-binding regions of antibodies or binding siteson substrate molecules. Since it is the interactive capacity and natureof a protein that defines that protein's biological functional activity,certain amino acid substitutions can be made in a protein sequence, andits underlying DNA coding sequence, and nevertheless obtain a proteinwith like properties. It is thus contemplated by the inventors thatvarious changes may be made in the DNA sequences of genes withoutappreciable loss of their biological utility or activity, as discussedbelow. Table 1 shows the codons that encode particular amino acids.

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982). It is accepted thatthe relative hydropathic character of the amino acid contributes to thesecondary structure of the resultant protein, which in turn defines theinteraction of the protein with other molecules, for example, enzymes,substrates, receptors, DNA, antibodies, antigens, and the like.

Each amino acid has been assigned a hydropathic index on the basis oftheir hydrophobicity and charge characteristics (Kyte and Doolittle,1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9);alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9), and arginine (−4.5).

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, i.e., still obtaina biological functionally equivalent protein. In making such changes,the substitution of amino acids whose hydropathic indices are within ±2is preferred, those which are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101, incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biologicalproperty of the protein. As detailed in U.S. Pat. No. 4,554,101, thefollowing hydrophilicity values have been assigned to amino acidresidues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate(+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine(0), threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine*−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine(−1.8), isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5);tryptophan (−3.4).

It is understood that an amino acid can be substituted for anotherhaving a similar hydrophilicity value and still obtain a biologicallyequivalent and immunologically equivalent protein. In such changes, thesubstitution of amino acids whose hydrophilicity values are within ±2 ispreferred, those that are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

As outlined above, amino acid substitutions are generally based on therelative similarity of the amino acid side-chain substituents, forexample, their hydrophobicity, hydrophilicity, charge, size, and thelike. Exemplary substitutions that take various of the foregoingcharacteristics into consideration are well known to those of skill inthe art and include: arginine and lysine; glutamate and aspartate;serine and threonine; glutamine and asparagine; and valine, leucine andisoleucine.

Another embodiment for the preparation of polypeptides according to theinvention is the use of peptide mimetics. Mimetics arepeptide-containing molecules that mimic elements of protein secondarystructure (Johnson et al, 1993). The underlying rationale behind the useof peptide mimetics is that the peptide backbone of proteins existschiefly to orient amino acid side chains in such a way as to facilitatemolecular interactions, such as those of antibody and antigen. A peptidemimetic is expected to permit molecular interactions similar to thenatural molecule. These principles may be used, in conjunction with theprinciples outline above, to engineer second generation molecules havingmany of the natural properties of MURF-1, MURF-2 and MURF-3, but withaltered and even improved characteristics.

D. Domain Switching

As described in the examples, the present inventors have identifiedmurine MURF-1, MURF-2 and MURF-3. Given the homology with other Midlineproteins, an interesting series of mutants can be created bysubstituting homologous regions of various proteins. This is known, incertain contexts, as “domain switching.”

Domain switching involves the generation of chimeric molecules usingdifferent but, in this case, related polypeptides. By comparing variousMidline proteins, one can make predictions as to the functionallysignificant regions of these molecules. It is possible, then, to switchrelated domains of these molecules in an effort to determine thecriticality of these regions to MURF-1, MURF-2 and MURF-3 function.These molecules may have additional value in that these “chimeras” canbe distinguished from natural molecules, while possibly providing thesame function.

Particular structural aspects of MURF-1 that provide fertile ground fordomain switching experiments are a RING finger domain (residues 26-81),a B-box (residues 126-158), a leucine-rich coiled-coil (residues212-253) and an acid region (residues 335-366). These domains may besubstituted for related domains of Midline proteins.

E. Fusion Proteins

A specialized kind of insertional variant is the fusion protein. Thismolecule generally has all or a substantial portion of the nativemolecule, linked at the N- or C-terminus, to all or a portion of asecond polypeptide. For example, fusions typically employ leadersequences from other species to permit the recombinant expression of aprotein in a heterologous host. Another useful fusion includes theaddition of a immunologically active domain, such as an antibodyepitope, to facilitate purification of the fusion protein. Inclusion ofa cleavage site at or near the fusion junction will facilitate removalof the extraneous polypeptide after purification. Other useful fusionsinclude linking of functional domains, such as active sites fromenzymes, glycosylation domains, cellular targeting signals ortransmembrane regions.

F. Purification of Proteins

It will be desirable to purify MURF-1, MURF-2, MURF-3 or variantsthereof. Protein purification techniques are well known to those ofskill in the art. These techniques involve, at one level, the crudefractionation of the cellular milieu to polypeptide and non-polypeptidefractions. Having separated the polypeptide from other proteins, thepolypeptide of interest may be further purified using chromatographicand electrophoretic techniques to achieve partial or completepurification (or purification to homogeneity). Analytical methodsparticularly suited to the preparation of a pure peptide areion-exchange chromatography, exclusion chromatography; polyacrylamidegel electrophoresis; isoelectric focusing. A particularly efficientmethod of purifying peptides is fast protein liquid chromatography oreven HPLC.

Certain aspects of the present invention concern the purification, andin particular embodiments, the substantial purification, of an encodedprotein or peptide. The term “purified protein or peptide” as usedherein, is intended to refer to a composition, isolatable from othercomponents, wherein the protein or peptide is purified to any degreerelative to its naturally-obtainable state. A purified protein orpeptide therefore also refers to a protein or peptide, free from theenvironment in which it may naturally occur.

Generally, “purified” will refer to a protein or peptide compositionthat has been subjected to fractionation to remove various othercomponents, and which composition substantially retains its expressedbiological activity. Where the term “substantially purified” is used,this designation will refer to a composition in which the protein orpeptide forms the major component of the composition, such asconstituting about 50%, about 60%, about 70%, about 80%, about 90%,about 95% or more of the proteins in the composition.

Various methods for quantifying the degree of purification of theprotein or peptide will be known to those of skill in the art in lightof the present disclosure. These include, for example, determining thespecific activity of an active fraction, or assessing the amount ofpolypeptides within a fraction by SDS/PAGE analysis. A preferred methodfor assessing the purity of a fraction is to calculate the specificactivity of the fraction, to compare it to the specific activity of theinitial extract, and to thus calculate the degree of purity, hereinassessed by a “-fold purification number.” The actual units used torepresent the amount of activity will, of course, be dependent upon theparticular assay technique chosen to follow the purification and whetheror not the expressed protein or peptide exhibits a detectable activity.

Various techniques suitable for use in protein purification will be wellknown to those of skill in the art. These include, for example,precipitation with ammonium sulphate, PEG, antibodies and the like or byheat denaturation, followed by centrifugation; chromatography steps suchas ion exchange, gel filtration, reverse phase, hydroxylapatite andaffinity chromatography; isoelectric focusing; gel electrophoresis; andcombinations of such and other techniques. As is generally known in theart, it is believed that the order of conducting the variouspurification steps may be changed, or that certain steps may be omitted,and still result in a suitable method for the preparation of asubstantially purified protein or peptide.

There is no general requirement that the protein or peptide always beprovided in their most purified state. Indeed, it is contemplated thatless substantially purified products will have utility in certainembodiments. Partial purification may be accomplished by using fewerpurification steps in combination, or by utilizing different forms ofthe same general purification scheme. For example, it is appreciatedthat a cation-exchange column chromatography performed utilizing an HPLCapparatus will generally result in a greater “-fold” purification thanthe same technique utilizing a low pressure chromatography system.Methods exhibiting a lower degree of relative purification may haveadvantages in total recovery of protein product, or in maintaining theactivity of an expressed protein.

It is known that the migration of a polypeptide can vary, sometimessignificantly, with different conditions of SDS/PAGE (Capaldi et al.,1977). It will therefore be appreciated that under differingelectrophoresis conditions, the apparent molecular weights of purifiedor partially purified expression products may vary.

High Performance Liquid Chromatography (HPLC) is characterized by a veryrapid separation with extraordinary resolution of peaks. This isachieved by the use of very fine particles and high pressure to maintainan adequate flow rate. Separation can be accomplished in a matter ofminutes, or at most an hour. Moreover, only a very small volume of thesample is needed because the particles are so small and close-packedthat the void volume is a very small fraction of the bed volume. Also,the concentration of the sample need not be very great because the bandsare so narrow that there is very little dilution of the sample.

Gel chromatography, or molecular sieve chromatography, is a special typeof partition chromatography that is based on molecular size. The theorybehind gel chromatography is that the column, which is prepared withtiny particles of an inert substance that contain small pores, separateslarger molecules from smaller molecules as they pass through or aroundthe pores, depending on their size. As long as the material of which theparticles are made does not adsorb the molecules, the sole factordetermining rate of flow is the size. Hence, molecules are eluted fromthe column in decreasing size, so long as the shape is relativelyconstant. Gel chromatography is unsurpassed for separating molecules ofdifferent size because separation is independent of all other factorssuch as pH, ionic strength, temperature, etc. There also is virtually noadsorption, less zone spreading and the elution volume is related in asimple matter to molecular weight.

Affinity Chromatography is a chromatographic procedure that relies onthe specific affinity between a substance to be isolated and a moleculethat it can specifically bind to. This is a receptor-ligand typeinteraction. The column material is synthesized by covalently couplingone of the binding partners to an insoluble matrix. The column materialis then able to specifically adsorb the substance from the solution.Elution occurs by changing the conditions to those in which binding willnot occur (alter pH, ionic strength, temperature, etc.).

A particular type of affinity chromatography useful in the purificationof carbohydrate containing compounds is lectin affinity chromatography.Lectins are a class of substances that bind to a variety ofpolysaccharides and glycoproteins. Lectins are usually coupled toagarose by cyanogen bromide. Conconavalin A coupled to Sepharose was thefirst material of this sort to be used and has been widely used in theisolation of polysaccharides and glycoproteins other lectins that havebeen include lentil lectin, wheat germ agglutinin which has been usefulin the purification of N-acetyl glucosaminyl residues and Helix pomatialectin. Lectins themselves are purified using affinity chromatographywith carbohydrate ligands. Lactose has been used to purify lectins fromcastor bean and peanuts; maltose has been useful in extracting lectinsfrom lentils and jack bean; N-acetyl-D galactosamine is used forpurifying lectins from soybean; N-acetyl glucosaminyl binds to lectinsfrom wheat germ; D-galactosamine has been used in obtaining lectins fromclams and L-fucose will bind to lectins from lotus.

The matrix should be a substance that itself does not adsorb moleculesto any significant extent and that has a broad range of chemical,physical and thermal stability. The ligand should be coupled in such away as to not affect its binding properties. The ligand should alsoprovide relatively tight binding. And it should be possible to elute thesubstance without destroying the sample or the ligand. One of the mostcommon forms of affinity chromatography is immunoaffinitychromatography. The generation of antibodies that would be suitable foruse in accord with the present invention is discussed below.

G. Synthetic Peptides

The present invention also describes smaller MURF-related peptides foruse in various embodiments of the present invention. Because of theirrelatively small size, the peptides of the invention can also besynthesized in solution or on a solid support in accordance withconventional techniques. Various automatic synthesizers are commerciallyavailable and can be used in accordance with known protocols. See, forexample, Stewart and Young, (1984); Tam et al., (1983); Merrifield,(1986); and Barany and Merrifield (1979), each incorporated herein byreference. Short peptide sequences, or libraries of overlappingpeptides, usually from about 6 up to about 35 to 50 amino acids, whichcorrespond to the selected regions described herein, can be readilysynthesized and then screened in screening assays designed to identifyreactive peptides. Alternatively, recombinant DNA technology may beemployed wherein a nucleotide sequence which encodes a peptide of theinvention is inserted into an expression vector, transformed ortransfected into an appropriate host cell and cultivated underconditions suitable for expression.

H. Antigen Compositions

The present invention also provides for the use of MURF-1, MURF-2 andMURF-3 proteins or peptides as antigens for the immunization of animalsrelating to the production of antibodies. It is envisioned that MURF-1,MURF-2, MURF-3, or portions thereof, will be coupled, bonded, bound,conjugated or chemically-linked to one or more agents via linkers,polylinkers or derivatized amino acids. This may be performed such thata bispecific or multivalent composition or vaccine is produced. It isfurther envisioned that the methods used in the preparation of thesecompositions will be familiar to those of skill in the art and should besuitable for administration to animals, i.e., pharmaceuticallyacceptable. Preferred agents are the carriers are keyhole limpethemocyannin (KLH) or bovine serum albumin (BSA).

III. Nucleic Acids

The present invention also provides, in another embodiment, genesencoding MURF-1, MURF-2 and MURF-3. Genes for murine MURF-1, MURF-2 andMURF-3 have been identified. The present invention is not limited inscope to these genes, however, as one of ordinary skill in the could,using these nucleic acids, readily identify related homologs in variousother species (e.g., rat, rabbit, dog, monkey, gibbon, human, chimp,ape, baboon, cow, pig, horse, sheep, cat and other species).

In addition, it should be clear that the present invention is notlimited to the specific nucleic acids disclosed herein. As discussedbelow, a “MURF gene” may contain a variety of different bases and yetstill produce a corresponding polypeptide that is functionallyindistinguishable, and in some cases structurally, from the human andmouse genes disclosed herein.

Similarly, any reference to a nucleic acid should be read asencompassing a host cell containing that nucleic acid and, in somecases, capable of expressing the product of that nucleic acid. Inaddition to therapeutic considerations, cells expressing nucleic acidsof the present invention may prove useful in the context of screeningfor agents that induce, repress, inhibit, augment, interfere with,block, abrogate, stimulate or enhance the function of MURF-1, MURF-2 orMURF-3.

A. Nucleic Acids Encoding MURFs

Nucleic acids according to the present invention may encode an entireMURF-1, MURF-2 or MURF-3 gene, a domain of MURF-1, MURF-2 or MURF-3, orany other fragment of MURF-1, MURF-2 or MURF-3 as set forth herein. Thenucleic acid may be derived from genomic DNA, i.e., cloned directly fromthe genome of a particular organism. In preferred embodiments, however,the nucleic acid would comprise complementary DNA (cDNA). Alsocontemplated is a cDNA plus a natural intron or an intron derived fromanother gene; such engineered molecules are sometime referred to as“mini-genes.” At a minimum, these and other nucleic acids of the presentinvention may be used as molecular weight standards in, for example, gelelectrophoresis.

The term “cDNA” is intended to refer to DNA prepared using messenger RNA(mRNA) as template. The advantage of using a cDNA, as opposed to genomicDNA or DNA polymerized from a genomic, non- or partially-processed RNAtemplate, is that the cDNA primarily contains coding sequences of thecorresponding protein. There may be times when the full or partialgenomic sequence is preferred, such as where the non-coding regions arerequired for optimal expression or where non-coding regions such asintrons are to be targeted in an antisense strategy.

It also is contemplated that a given MURF from a given species may berepresented by natural variants that have slightly different nucleicacid sequences but, nonetheless, encode the same protein (see Table 1below).

As used in this application, the term “a nucleic acid encoding a MURF”refers to a nucleic acid molecule that has been isolated free of totalcellular nucleic acid. In preferred embodiments, the invention concernsa nucleic acid sequence essentially as set forth in SEQ ID NOS: 1 and 3.The term “as set forth in SEQ ID NOS: 1 or 3 or 5” means that thenucleic acid sequence substantially corresponds to a portion of SEQ IDNO:1, 3 or 5 The term “functionally equivalent codon” is used herein torefer to codons that encode the same amino acid, such as the six codonsfor arginine or serine (Table 1, below), and also refers to codons thatencode biologically equivalent amino acids, as discussed in thefollowing pages.

TABLE 1 Amino Acids Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys CUGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAGPhenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine HisH CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine LeuL UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAUProline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGAAGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr TACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGGTyrosine Tyr Y UAC UAU

Allowing for the degeneracy of the genetic code, sequences that have atleast about 50%, usually at least about 60%, more usually about 70%,most usually about 80%, preferably at least about 90% and mostpreferably about 95% of nucleotides that are identical to thenucleotides of SEQ ID NOS. 1 or 3 or 5 are contemplated. Sequences thatare essentially the same as those set forth in SEQ ID NOS:1, 3 and 5 mayalso be functionally defined as sequences that are capable ofhybridizing to a nucleic acid segment containing the complement of SEQID NOS: 1, 3 and 5 under standard conditions.

The DNA segments of the present invention include those encodingbiologically functional equivalent MURF proteins and peptides, asdescribed above. Such sequences may arise as a consequence of codonredundancy and amino acid functional equivalency that are known to occurnaturally within nucleic acid sequences and the proteins thus encoded.Alternatively, functionally equivalent proteins or peptides may becreated via the application of recombinant DNA technology, in whichchanges in the protein structure may be engineered, based onconsiderations of the properties of the amino acids being exchanged.Changes designed by man may be introduced through the application ofsite-directed mutagenesis techniques or may be introduced randomly andscreened later for the desired function, as described below.

B. Oligonucleotide Probes and Primers

Naturally, the present invention also encompasses DNA segments that arecomplementary, or essentially complementary, to the sequence set forthin SEQ ID NOS: 1, 3 and 5. Nucleic acid sequences that are“complementary” are those that are capable of base-pairing according tothe standard Watson-Crick complementary rules. As used herein, the term“complementary sequences” means nucleic acid sequences that aresubstantially complementary, as may be assessed by the same nucleotidecomparison set forth above, or as defined as being capable ofhybridizing to the nucleic acid segment of SEQ ID NOS: 1, 3 and 5 underrelatively stringent conditions such as those described herein. Suchsequences may encode the entire MURF proteins or functional ornon-functional fragments thereof.

Alternatively, the hybridizing segments may be shorter oligonucleotides.Sequences of 17 bases long should occur only once in the human genomeand, therefore, suffice to specify a unique target sequence. Althoughshorter oligomers are easier to make and increase in vivo accessibility,numerous other factors are involved in determining the specificity ofhybridization. Both binding affinity and sequence specificity of anoligonucleotide to its complementary target increases with increasinglength. It is contemplated that exemplary oligonucleotides of 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, 95, 100 or more base pairs will be used,although others are contemplated. Longer polynucleotides encoding 250,500, 1000, 1212, 1500, 2000, 2500, 3000 or 5000 bases and longer arecontemplated as well. Such oligonucleotides will find use, for example,as probes in Southern and Northern blots and as primers in amplificationreactions.

Suitable hybridization conditions will be well known to those of skillin the art. In certain applications, for example, substitution of aminoacids by site-directed mutagenesis, it is appreciated that lowerstringency conditions are required. Under these conditions,hybridization may occur even though the sequences of probe and targetstrand are not perfectly complementary, but are mismatched at one ormore positions. Conditions may be rendered less stringent by increasingsalt concentration and decreasing temperature. For example, a mediumstringency condition could be provided by about 0.1 to 0.25 M NaCl attemperatures of about 37° C. to about 55° C., while a low stringencycondition could be provided by about 0.15 M to about 0.9 M salt, attemperatures ranging from about 20° C. to about 55° C. Thus,hybridization conditions can be readily manipulated, and thus willgenerally be a method of choice depending on the desired results

In other embodiments, hybridization may be achieved under conditions of,for example, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl₂, 10 mMdithiothreitol, at temperatures between approximately 20° C. to about37° C. Other hybridization conditions utilized could includeapproximately 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 μM MgCl₂, attemperatures ranging from approximately 40° C. to about 72° C. Formamideand SDS also may be used to alter the hybridization conditions.

One method of using probes and primers of the present invention is inthe search for genes related to MURF-1, MURF-2 or MURF-3 or, moreparticularly, homologs of MURF-1, MURF-2 or MURF-3 from other species.Normally, the target DNA will be a genomic or cDNA library, althoughscreening may involve analysis of RNA molecules. By varying thestringency of hybridization, and the region of the probe, differentdegrees of homology may be discovered.

Another way of exploiting probes and primers of the present invention isin site-directed, or site-specific mutagenesis. Site-specificmutagenesis is a technique useful in the preparation of individualpeptides, or biologically functional equivalent proteins or peptides,through specific mutagenesis of the underlying DNA. The techniquefurther provides a ready ability to prepare and test sequence variants,incorporating one or more of the foregoing considerations, byintroducing one or more nucleotide sequence changes into the DNA.Site-specific mutagenesis allows the production of mutants through theuse of specific oligonucleotide sequences which encode the DNA sequenceof the desired mutation, as well as a sufficient number of adjacentnucleotides, to provide a primer sequence of sufficient size andsequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Typically, a primer of about 17 to 25nucleotides in length is preferred, with about 5 to 10 residues on bothsides of the junction of the sequence being altered.

The technique typically employs a bacteriophage vector that exists inboth a single-stranded and double-stranded form. Typical vectors usefulin site-directed mutagenesis include vectors such as the M13 phage.These phage vectors are commercially available and their use isgenerally well known to those skilled in the art. Double-strandedplasmids are also routinely employed in site directed mutagenesis, whicheliminates the step of transferring the gene of interest from a phage toa plasmid.

In general, site-directed mutagenesis is performed by first obtaining asingle-stranded vector, or melting of two strands of a double-strandedvector which includes within its sequence a DNA sequence encoding thedesired protein. An oligonucleotide primer bearing the desired mutatedsequence is synthetically prepared. This primer is then annealed withthe single-stranded DNA preparation, taking into account the degree ofmismatch when selecting hybridization conditions, and subjected to DNApolymerizing enzymes such as E. coli polymerase I Klenow fragment, inorder to complete the synthesis of the mutation-bearing strand. Thus, aheteroduplex is formed wherein one strand encodes the originalnon-mutated sequence and the second strand bears the desired mutation.This heteroduplex vector is then used to transform appropriate cells,such as E. coli cells, and clones are selected that include recombinantvectors bearing the mutated sequence arrangement.

The preparation of sequence variants of the selected gene usingsite-directed mutagenesis is provided as a means of producingpotentially useful species and is not meant to be limiting, as there areother ways in which sequence variants of genes may be obtained. Forexample, recombinant vectors encoding the desired gene may be treatedwith mutagenic agents, such as hydroxylamine, to obtain sequencevariants.

C. Antisense Constructs

Antisense methodology takes advantage of the fact that nucleic acidstend to pair with “complementary” sequences. By complementary, it ismeant that polynucleotides are those which are capable of base-pairingaccording to the standard Watson-Crick complementarity rules. That is,the larger purines will base pair with the smaller pyrimidines to formcombinations of guanine paired with cytosine (G:C) and adenine pairedwith either thymine (A:T) in the case of DNA, or adenine paired withuracil (A:U) in the case of RNA. Inclusion of less common bases such asinosine, 5-methylcytosine, 6-methyladenine, hypoxanthine and others inhybridizing sequences does not interfere with pairing.

Targeting double-stranded (ds) DNA with polynucleotides leads totriple-helix formation; targeting RNA will lead to double-helixformation. Antisense polynucleotides, when introduced into a targetcell, specifically bind to their target polynucleotide and interferewith transcription, RNA processing, transport, translation and/orstability. Antisense RNA constructs, or DNA encoding such antisenseRNA's, may be employed to inhibit gene transcription or translation orboth within a host cell, either in vitro or in vivo, such as within ahost animal, including a human subject.

Antisense constructs may be designed to bind to the promoter and othercontrol regions, exons, introns or even exon-intron boundaries of agene. It is contemplated that the most effective antisense constructswill include regions complementary to intron/exon splice junctions.Thus, it is proposed that a preferred embodiment includes an antisenseconstruct with complementarity to regions within 50-200 bases of anintron-exon splice junction. It has been observed that some exonsequences can be included in the construct without seriously affectingthe target selectivity thereof. The amount of exonic material includedwill vary depending on the particular exon and intron sequences used.One can readily test whether too much exon DNA is included simply bytesting the constructs in vitro to determine whether normal cellularfunction is affected or whether the expression of related genes havingcomplementary sequences is affected.

As stated above, “complementary” or “antisense” means polynucleotidesequences that are substantially complementary over their entire lengthand have very few base mismatches. For example, sequences of fifteenbases in length may be termed complementary when they have complementarynucleotides at thirteen or fourteen positions. Naturally, sequenceswhich are completely complementary will be sequences which are entirelycomplementary throughout their entire length and have no basemismatches. Other sequences with lower degrees of homology also arecontemplated. For example, an antisense construct which has limitedregions of high homology, but also contains a non-homologous region(e.g., ribozyme; see below) could be designed. These molecules, thoughhaving less than 50% homology, would bind to target sequences underappropriate conditions.

It may be advantageous to combine portions of genomic DNA with cDNA orsynthetic sequences to generate specific constructs. For example, wherean intron is desired in the ultimate construct, a genomic clone willneed to be used. The cDNA or a synthesized polynucleotide may providemore convenient restriction sites for the remaining portion of theconstruct and, therefore, would be used for the rest of the sequence.

D. Ribozymes

Although proteins traditionally have been used for catalysis of nucleicacids, another class of macromolecules has emerged as usefull in thisendeavor. Ribozymes are RNA-protein complexes that cleave nucleic acidsin a site-specific fashion. Ribozymes have specific catalytic domainsthat possess endonuclease activity (Kim and Cook, 1987; Gerlach et al.1987; Forster and Symons, 1987). For example, a large number ofribozymes accelerate phosphoester transfer reactions with a high degreeof specificity, often cleaving only one of several phosphoesters in anoligonucleotide substrate (Cook et al., 1981; Michel and Westhof, 1990;Reinhold-Hurek and Shub, 1992). This specificity has been attributed tothe requirement that the substrate bind via specific base-pairinginteractions to the internal guide sequence (“IGS”) of the ribozymeprior to chemical reaction.

Ribozyme catalysis has primarily been observed as part ofsequence-specific cleavage/ligation reactions involving nucleic acids(Joyce, 1989; Cook et al., 1981). For example, U.S. Pat. No. 5,354,855reports that certain ribozymes can act as endonucleases with a sequencespecificity greater than that of known ribonucleases and approachingthat of the DNA restriction enzymes. Thus, sequence-specificribozyme-mediated inhibition of gene expression may be particularlysuited to therapeutic applications (Scanlon et al., 1991; Sarver et al.1990). Recently, it was reported that ribozymes elicited genetic changesin some cells lines to which they were applied, the altered genesincluded the oncogenes H-ras, c-los and genes of HIV. Most of this workinvolved the modification of a target mRNA, based on a specific mutantcodon that is cleaved by a specific ribozyme.

E. Vectors for Cloning, Gene Transfer and Expression

Within certain embodiments expression vectors are employed to express aMURF polypeptide product, which can then be purified and, for example,be used to vaccinate animals to generate antisera or monoclonal antibodywith which further studies may be conducted. In other embodiments, theexpression vectors are used in gene therapy. Expression requires thatappropriate signals be provided in the vectors, and which includevarious regulatory elements, such as enhancers/promoters from both viraland mammalian sources that drive expression of the genes of interest inhost cells. Elements designed to optimize messenger RNA stability andtranslatability in host cells also are defined. The conditions for theuse of a number of dominant drug selection markers for establishingpermanent, stable cell clones expressing the products are also provided,as is an element that links expression of the drug selection markers toexpression of the polypeptide.

(i) Regulatory Elements

Throughout this application, the term “expression construct” is meant toinclude any type of genetic construct containing a nucleic acid codingfor a gene product in which part or all of the nucleic acid encodingsequence is capable of being transcribed. The transcript may betranslated into a protein, but it need not be. In certain embodiments,expression includes both transcription of a gene and translation of mRNAinto a gene product. In other embodiments, expression only includestranscription of the nucleic acid encoding a gene of interest.

In preferred embodiments, the nucleic acid encoding a gene product isunder transcriptional control of a promoter. A “promoter” refers to aDNA sequence recognized by the synthetic machinery of the cell, orintroduced synthetic machinery, required to initiate the specifictranscription of a gene. The phrase “under transcriptional control”means that the promoter is in the correct location and orientation inrelation to the nucleic acid to control RNA polymerase initiation andexpression of the gene.

The term promoter will be used here to refer to a group oftranscriptional control modules that are clustered around the initiationsite for RNA polymerase II. Much of the thinking about how promoters areorganized derives from analyses of several viral promoters, includingthose for the HSV thymidine kinase (tk) and SV40 early transcriptionunits. These studies, augmented by more recent work, have shown thatpromoters are composed of discrete functional modules, each consistingof approximately 7-20 bp of DNA, and containing one or more recognitionsites for transcriptional activator or repressor proteins.

At least one module in each promoter functions to position the startsite for RNA synthesis. The best known example of this is the TATA box,but in some promoters lacking a TATA box, such as the promoter for themammalian terminal deoxynucleotidyl transferase gene and the promoterfor the SV40 late genes, a discrete element overlying the start siteitself helps to fix the place of initiation.

Additional promoter elements regulate the frequency of transcriptionalinitiation. Typically, these are located in the region 30-110 bpupstream of the start site, although a number of promoters have recentlybeen shown to contain functional elements downstream of the start siteas well. The spacing between promoter elements frequently is flexible,so that promoter function is preserved when elements are inverted ormoved relative to one another. In the tk promoter, the spacing betweenpromoter elements can be increased to 50 bp apart before activity beginsto decline. Depending on the promoter, it appears that individualelements can unction either co-operatively or independently to activatetranscription.

In various embodiments, the human cytomegalovirus (CMV) immediate earlygene promoter, the SV40 early promoter, the Rous sarcoma virus longterminal repeat, rat insulin promoter and glyceraldehyde-3-phosphatedehydrogenase can be used to obtain high-level expression of the codingsequence of interest. The use of other viral or mammalian cellular orbacterial phage promoters which are well-known in the art to achieveexpression of a coding sequence of interest is contemplated as well,provided that the levels of expression are sufficient for a givenpurpose.

By employing a promoter with well-known properties, the level andpattern of expression of the protein of interest following transfectionor transformation can be optimized. Further, selection of a promoterthat is regulated in response to specific physiologic signals can permitinducible expression of the gene product. Tables 2 and 3 list severalregulatory elements that may be employed, in the context of the presentinvention, to regulate the expression of the gene of interest. This listis not intended to be exhaustive of all the possible elements involvedin the promotion of gene expression but, merely, to be exemplary thereof

Enhancers are genetic elements that increase transcription from apromoter located at a distant position on the same molecule of DNA.Enhancers are organized much like promoters. That is, they are composedof many individual elements, each of which binds to one or moretranscriptional proteins.

The basic distinction between enhancers and promoters is operational. Anenhancer region as a whole must be able to stimulate transcription at adistance; this need not be true of a promoter region or its componentelements. On the other hand, a promoter must have one or more elementsthat direct initiation of RNA synthesis at a particular site and in aparticular orientation, whereas enhancers lack these specificities.Promoters and enhancers are often overlapping and contiguous, oftenseeming to have a very similar modular organization.

Below is a list of viral promoters, cellular promoters/enhancers andinducible promoters/enhancers that could be used in combination with thenucleic acid encoding a gene of interest in an expression construct(Table 2 and Table 3). Additionally, any promoter/enhancer combination(as per the Eukaryotic Promoter Data Base EPDB) could also be used todrive expression of the gene. Eukaryotic cells can support cytoplasmictranscription from certain bacterial promoters if the appropriatebacterial polymerase is provided, either as part of the delivery complexor as an additional genetic expression construct.

TABLE 2 Promoter and/or Enhancer Promoter/Enhancer ReferencesImmunoglobulin Banerji et al., 1983; Gilles et al., 1983; Heavy ChainGrosschedl et al., 1985; Atchinson et al., 1986, 1987; Imler et al.,1987; Weinberger et al., 1984; Kiledjian et al., 1988; Porton et al.;1990 Immunoglobulin Queen et al., 1983; Picard et al., 1984 Light ChainT-Cell Receptor Luria et al., 1987; Winoto et al., 1989; Redondo et al.;1990 HLA DQ a and/or Sullivan et al., 1987 DQ β β-Interferon Goodbournet al., 1986; Fujita et al., 1987; Goodbourn et al., 1988 Interleukin-2Greene et al., 1989 Interleukin-2 Receptor Greene et al., 1989; Lin etal., 1990 MHC Class II 5 Koch et al., 1989 MHC Class II Sherman et al.,1989 HLA-DRa β-Actin Kawamoto et al., 1988; Ng et al.; 1989 MuscleCreatine Jaynes et al., 1988; Horlick et al., 1989; Johnson Kinase (MCK)et al., 1989 Prealbumin Costa et al., 1988 (Transthyretin) Elastase IOrnitz et al., 1987 Metallothionein Karin et al., 1987; Culotta et al.,1989 (MTII) Collagenase Pinkert et al., 1987; Angel et al., 1987aAlbumin Pinkert et al., 1987; Tronche et al., 1989, 1990 α-FetoproteinGodbout et al., 1988; Campere et al., 1989 t-Globin Bodine et al., 1987;Perez-Stable et al., 1990 β-Globin Trudel et al., 1987 c-fos Cohen etal., 1987 c-HA-ras Triesman, 1986; Deschamps et al., 1985 Insulin Edlundet al., 1985 Neural Cell Hirsh et al., 1990 Adhesion Molecule (NCAM)α₁-Antitrypain Latimer et al., 1990 H2B (TH2B) Histone Hwang et al.,1990 Mouse and/or Type I Ripe et al., 1989 Collagen Glucose-RegulatedChang et al., 1989 Proteins (GRP94 and GRP78) Rat Growth Hormone Larsenet al., 1986 Human Serum Edbrooke et al., 1989 Amyloid A (SAA) TroponinI (TN I) Yutzey et al., 1989 Platelet-Derived Pech et al., 1989 GrowthFactor (PDGF) Duchenne Muscular Klamut et al., 1990 Dystrophy SV40Banerji et al., 1981; Moreau et al., 1981; Sleigh et al., 1985; Firak etal., 1986; Herr et al., 1986; Imbra et al., 1986; Kadesch et al., 1986;Wang et al., 1986; Ondek et al., 1987; Kuhl et al., 1987; Schaffner etal., 1988 Polyoma Swartzendruber et al., 1975; Vasseur et al., 1980;Katinka et al., 1980, 1981; Tyndell et al., 1981; Dandolo et al., 1983;de Villiers et al., 1984; Hen et al., 1986; Satake et al., 1988;Campbell and/or Villarreal, 1988 Retroviruses Kriegler et al., 1982,1983; Levinson et al., 1982; Kriegler et al., 1983, 1984a, b, 1988;Bosze et al., 1986; Miksicek et al., 1986; Celander et al., 1987;Thiesen et al., 1988; Celander et al., 1988; Choi et al., 1988; Reismanet al., 1989 Papilloma Virus Campo et al., 1983; Lusky et al., 1983;Spandidos and/or Wilkie, 1983; Spalholz et al., 1985; Lusky et al.,1986; Cripe et al., 1987; Gloss et al., 1987; Hirochika et al., 1987;Stephens et al., 1987; Glue et al., 1988 Hepatitis B Virus Bulla et al.,1986; Jameel et al., 1986; Shaul et al., 1987; Spandau et al., 1988;Vannice et al., 1988 Human Muesing et al., 1987; Hauber et al., 1988;Immunodeficiency Jakobovits et al., 1988; Feng et al., 1988; TakebeVirus et al., 1988; Rosen et al., 1988; Berkhout et al., 1989; Laspia etal., 1989; Sharp et al., 1989; Braddock et al., 1989 CytomegalovirusWeber et al., 1984; Boshart et al., 1985; Foecking (CMV) et al., 1986Gibbon Ape Holbrook et al., 1987; Quinn et al., 1989 Leukemia Virus

TABLE 3 Inducible Elements Element Inducer References MT II PhorbolEster (TFA) Palmiter et al., 1982, Heavy metals Haslinger et al., 1985;Searle et al., 1985; Stuart et al., 1985; Imagawa et al., 1987, Karin etal., 1987; Angel et al., 1987b; McNeall et al., 1989 MMTV (mouseGlucocorticoids Huang et al., 1981; Lee mammary tumor virus) et al.,1981; Majors et al., 1983; Chandler et al., 1983; Lee et al., 1984;Ponta et al., 1985; Sakai et al., 1988 β-Interferon poly(rI)x Tavernieret al., 1983 poly(rc) Adenovirus 5 E2 E1A Imperiale et al., 1984Collagenase Phorbol Ester (TPA) Angel et al., 1987a Stromelysin PhorbolEster (TPA) Angel et al., 1987b SV40 Phorbol Ester (TPA) Angel et al.,1987b Murine MX Gene Interferon, New- Hug et al., 1988 castle DiseaseVirus GRP78 Gene A23187 Resendez et al., 1988 α-2-Macroglobulin IL-6Kunz et al., 1989 Vimentin Serum Rittling et al., 1989 MHC Class IInterferon Blanar et al., 1989 Gene H-2κb HSP70 E1A, SV40 Large T Tayloret al., 1989, Antigen 1990a, 1990b Proliferin Phorbol Ester-TPA Mordacqet al., 1989 Tumor Necrosis Factor PMA Hensel et al., 1989 ThyroidStimulating Thyroid Hormone Chatterjee et al., 1989 Hormone α Gene

Of particular interest are muscle specific promoters, and moreparticularly, cardiac specific promoters. These include the myosin lightchain-2 promoter (Franz et al., 1994; Kelly et al. 1995), the alphaactin promoter (Moss et al., 1996), the troponin 1 promoter (Bhavsar etal, 1996); the Na⁺/Ca²⁺ exchanger promoter (Barnes et al. 1997), thedystrophin promoter (Kimura et al., 1997), the creatine kinase promoter(Ritchie, M. E., 1996), the alpha7 integrin promoter (Ziober & Kramer,1996), the brain natriuretic peptide promoter (LaPointe et al., 1996),the alpha B-crystallin/small heat shock protein promoter(Gopal-Srivastava, R., 1995), and alpha myosin heavy chain promoter(Yamauchi-Takihara et al., 1989) and the ANF promoter (LaPointe et al.,1988).

Where a cDNA insert is employed, one will typically desire to include apolyadenylation signal to effect proper polyadenylation of the genetranscript. The nature of the polyadenylation signal is not believed tobe crucial to the successful practice of the invention, and any suchsequence may be employed such as human growth hormone and SV40polyadenylation signals. Also contemplated as an element of theexpression cassette is a terminator. These elements can serve to enhancemessage levels and to minimize read through from the cassette into othersequences.

(ii) Selectable Markers

In certain embodiments of the invention, the cells contain nucleic acidconstructs of the present invention, a cell may be identified in vitroor in vivo by including a marker in the expression construct. Suchmarkers would confer an identifiable change to the cell permitting easyidentification of cells containing the expression construct. Usually theinclusion of a drug selection marker aids in cloning and in theselection of transformants, for example, genes that confer resistance toneomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol areuseful selectable markers. Alternatively, enzymes such as herpes simplexvirus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT)may be employed. Immunologic markers also can be employed. Theselectable marker employed is not believed to be important, so long asit is capable of being expressed simultaneously with the nucleic acidencoding a gene product. Further examples of selectable markers are wellknown to one of skill in the art.

(iii) Multigene Constructs and IRES

In certain embodiments of the invention, the use of internal ribosomebinding sites (IRES) elements are used to create multigene, orpolycistronic, messages. IRES elements are able to bypass the ribosomescanning model of 5′ methylated Cap dependent translation and begintranslation at internal sites (Pelletier and Sonenberg, 1988). IRESelements from two members of the picanovirus family (polio andencephalomyocarditis) have been described (Pelletier and Sonenberg,1988), as well an IRES from a mammalian message (Macejak and Sarnow,1991). IRES elements can be linked to heterologous open reading frames.Multiple open reading frames can be transcribed together, each separatedby an IRES, creating polycistronic messages. By virtue of the IRESelement, each open reading frame is accessible to ribosomes forefficient translation. Multiple genes can be efficiently expressed usinga single promoter/enhancer to transcribe a single message.

Any heterologous open reading frame can be linked to IRES elements. Thisincludes genes for secreted proteins, multi-subunit proteins, encoded byindependent genes, intracellular or membrane-bound proteins andselectable markers. In this way, expression of several proteins can besimultaneously engineered into a cell with a single construct and asingle selectable marker.

(iv) Delivery of Expression Vectors

There are a number of ways in which expression vectors may introducedinto cells. In certain embodiments of the invention, the expressionconstruct comprises a virus or engineered construct derived from a viralgenome. The ability of certain viruses to enter cells viareceptor-mediated endocytosis, to integrate into host cell genome andexpress viral genes stably and efficiently have made them attractivecandidates for the transfer of foreign genes into mammalian cells(Ridgeway, 1988; Nicolas and Rubenstein, 1988; Baichwal and Sugden,1986; Temin, 1986). The first viruses used as gene vectors were DNAviruses including the papovaviruses (simian virus 40, bovine papillomavirus, and polyoma) (Ridgeway, 1988; Baichwal and Sugden, 1986) andadenoviruses (Ridgeway, 1988; Baichwal and Sugden, 1986). These have arelatively low capacity for foreign DNA sequences and have a restrictedhost spectrum. Furthermore, their oncogenic potential and cytopathiceffects in permissive cells raise safety concerns. They can accommodateonly up to 8 kb of foreign genetic material but can be readilyintroduced in a variety of cell lines and laboratory animals (Nicolasand Rubenstein, 1988; Temin, 1986).

One of the preferred methods for in vivo delivery involves the use of anadenovirus expression vector. “Adenovirus expression vector” is meant toinclude those constructs containing adenovirus sequences sufficient to(a) support packaging of the construct and (b) to express an antisensepolynucleotide that has been cloned therein. In this context, expressiondoes not require that the gene product be synthesized.

The expression vector comprises a genetically engineered form ofadenovirus. Knowledge of the genetic organization of adenovirus, a 36kb, linear, double-stranded DNA virus, allows substitution of largepieces of adenoviral DNA with foreign sequences up to 7 kb (Grunhaus andHorwitz, 1992). In contrast to retrovirus, the adenoviral infection ofhost cells does not result in chromosomal integration because adenoviralDNA can replicate in an episomal manner without potential genotoxicity.Also, adenoviruses are structurally stable, and no genome rearrangementhas been detected after extensive amplification. Adenovirus can infectvirtually all epithelial cells regardless of their cell cycle stage. Sofar, adenoviral infection appears to be linked only to mild disease suchas acute respiratory disease in humans.

Adenovirus is particularly suitable for use as a gene transfer vectorbecause of its mid-sized genome, ease of manipulation, high titer, widetarget cell range and high infectivity. Both ends of the viral genomecontain 100-200 base pair inverted repeats (ITRs), which are ciselements necessary for viral DNA replication and packaging. The early(E) and late (L) regions of the genome contain different transcriptionunits that are divided by the onset of viral DNA replication. The E1region (E1A and E1B) encodes proteins responsible for the regulation oftranscription of the viral genome and a few cellular genes. Theexpression of the E2 region (E2A and E2B) results in the synthesis ofthe proteins for viral DNA replication. These proteins are involved inDNA replication, late gene expression and host cell shut-off (Renan,1990). The products of the late genes, including the majority of theviral capsid proteins, are expressed only after significant processingof a single primary transcript issued by the major late promoter (NLP).The MLP, (located at 16.8 m.u.) is particularly efficient during thelate phase of infection, and all the mRNA's issued from this promoterpossess a 5′-tripartite leader (TPL) sequence which makes them preferredmRNA's for translation.

In a current system, recombinant adenovirus is generated from homologousrecombination between shuttle vector and provirus vector. Due to thepossible recombination between two proviral vectors, wild-typeadenovirus may be generated from this process. Therefore, it is criticalto isolate a single clone of virus from an individual plaque and examineits genomic structure.

Generation and propagation of the current adenovirus vectors, which arereplication deficient, depend on a unique helper cell line, designated293, which was transformed from human embryonic kidney cells by Ad5 DNAfragments and constitutively expresses E1 proteins (Graham et al.,1977). Since the E3 region is dispensable from the adenovirus genome(Jones and Shenk, 1978), the current adenovirus vectors, with the helpof 293 cells, carry foreign DNA in either the E1, the D3 or both regions(Graham and Prevec, 1991). In nature, adenovirus can packageapproximately 105% of the wild-type genome (Ghosh-Choudhury et al.1987), providing capacity for about 2 extra kb of DNA. Combined with theapproximately 5.5 kb of DNA that is replaceable in the E1 and E3regions, the maximum capacity of the current adenovirus vector is under7.5 kb, or about 15% of the total length of the vector. More than 80% ofthe adenovirus viral genome remains in the vector backbone and is thesource of vector-borne cytotoxicity. Also, the replication deficiency ofthe E1-deleted virus is incomplete. For example, leakage of viral geneexpression has been observed with the currently available vectors athigh multiplicities of infection (MOI) (Mulligan, 1993).

Helper cell lines may be derived from human cells such as humanembryonic kidney cells, muscle cells, hematopoietic cells or other humanembryonic mesenchymal or epithelial cells. Alternatively, the helpercells may be derived from the cells of other mammalian species that arepermissive for human adenovirus. Such cells include, e.g., Vero cells orother monkey embryonic mesenchymal or epithelial cells. As stated above,the preferred helper cell line is 293.

Racher et al. (1995) disclosed improved methods for culturing 293 cellsand propagating adenovirus. In one format, natural cell aggregates aregrown by inoculating individual cells into 1 liter siliconized spinnerflasks (Techne, Cambridge, UK) containing 100-200 ml of medium.Following stirring at 40 rpm, the cell viability is estimated withtrypan blue. In another format, Fibra-Cel microcarriers (Bibby Sterlin,Stone, UK) (5 g/l) is employed as follows. A cell inoculum, resuspendedin 5 ml of medium, is added to the carrier (50 ml) in a 250 mlErlenmeyer flask and left stationary, with occasional agitation, for Ito 4 h. The medium is then replaced with 50 ml of fresh medium andshaking initiated. For virus production, cells are allowed to grow toabout 80% confluence, after which time the medium is replaced (to 25% ofthe final volume) and adenovirus added at an MOI of 0.05. Cultures areleft stationary overnight, following which the volume is increased to100% and shaking commenced for another 72 h.

Other than the requirement that the adenovirus vector be replicationdefective, or at least conditionally defective, the nature of theadenovirus vector is not believed to be crucial to the successfulpractice of the invention. The adenovirus may be of any of the 42different known serotypes or subgroups A-F. Adenovirus type 5 ofsubgroup C is the preferred starting material in order to obtain theconditional replication-defective adenovirus vector for use in thepresent invention. This is because Adenovirus type 5 is a humanadenovirus about which a great deal of biochemical and geneticinformation is known, and it has historically been used for mostconstructions employing adenovirus as a vector.

As stated above, the typical vector according to the present inventionis replication defective and will not have an adenovirus El region.Thus, it will be most convenient to introduce the polynucleotideencoding the gene of interest at the position from which the E1-codingsequences have been removed. However, the position of insertion of theconstruct within the adenovirus sequences is not critical to theinvention. The polynucleotide encoding the gene of interest may also beinserted in lieu of the deleted E3 region in E3 replacement vectors, asdescribed by Karlsson et al. (1986), or in the E4 region where a helpercell line or helper virus complements the E4 defect.

Adenovirus is easy to grow and manipulate and exhibits broad host rangein vitro and in vivo. This group of viruses can be obtained in hightiters, e.g., 10⁹-10¹² plaque-forming units per ml, and they are highlyinfective. The life cycle of adenovirus does not require integrationinto the host cell genome. The foreign genes delivered by adenovirusvectors are episomal and, therefore, have low genotoxicity to hostcells. No side effects have been reported in studies of vaccination withwild-type adenovirus (Couch et al., 1963; Top et al., 1971),demonstrating their safety and therapeutic potential as in vivo genetransfer vectors.

Adenovirus vectors have been used in eukaryotic gene expression (Levreroet al., 1991; Gomez-Foix et al., 1992) and vaccine development (Grunhausand Horwitz, 1992; Graham and Prevec, 1992). Recently, animal studiessuggested that recombinant adenovirus could be used for gene therapy(Stratford-Perricaudet and Perricaudet, 1991; Stratford-Perricaudet etal., 1990, Rich et al., 1993). Studies in administering recombinantadenovirus to different tissues include trachea instillation (Rosenfeldet al. 1991; Rosenfeld et al., 1992), muscle injection (Ragot et al.,1993), peripheral intravenous injections (Herz and Gerard, 1993) andstereotactic inoculation into the brain (Le Gal La Salle et al., 1993).

The retroviruses are a group of single-stranded RNA virusescharacterized by an ability to convert their RNA to double-stranded DNAin infected cells by a process of reverse-transcription (Coffin, 1990).The resulting DNA then stably integrates into cellular chromosomes as aprovirus and directs synthesis of viral proteins. The integrationresults in the retention of the viral gene sequences in the recipientcell and its descendants. The retroviral genome contains three genes,gag, pol, and env that code for capsid proteins, polymerase enzyme, andenvelope components, respectively. A sequence found upstream from thegag gene contains a signal for packaging of the genome into virions. Twolong terminal repeat (LTR) sequences are present at the 5′ and 3′ endsof the viral genome. These contain strong promoter and enhancersequences and are also required for integration in the host cell genome(Coffin, 1990).

In order to construct a retroviral vector, a nucleic acid encoding agene of interest is inserted into the viral genome in the place ofcertain viral sequences to produce a virus that isreplication-defective. In order to produce virions, a packaging cellline containing the gag, pol, and env genes but without the LTR andpackaging components is constructed (Mann et al., 1983). When arecombinant plasmid containing a cDNA, together with the retroviral LTRand packaging sequences is introduced into this cell line (by calciumphosphate precipitation for example), the packaging sequence allows theRNA transcript of the recombinant plasmid to be packaged into viralparticles, which are then secreted into the culture media (Nicolas andRubenstein, 1988; Temin, 1986; Mann et al., 1983). The media containingthe recombinant retroviruses is then collected, optionally concentrated,and used for gene transfer. Retroviral vectors are able to infect abroad variety of cell types. However, integration and stable expressionrequire the division of host cells (Paskind et al., 1975).

A novel approach designed to allow specific targeting of retrovirusvectors was recently developed based on the chemical modification of aretrovirus by the chemical addition of lactose residues to the viralenvelope. This modification could permit the specific infection ofhepatocytes via sialoglycoprotein receptors.

A different approach to targeting of recombinant retroviruses wasdesigned in which biotinylated antibodies against a retroviral envelopeprotein and against a specific cell receptor were used. The antibodieswere coupled via the biotin components by using streptavidin (Roux etal., 1989). Using antibodies against major histocompatibility complexclass I and class II antigens, they demonstrated the infection of avariety of human cells that bore those surface antigens with anecotropic virus in vitro (Roux et al., 1989).

There are certain limitations to the use of retrovirus vectors in allaspects of the present invention. For example, retrovirus vectorsusually integrate into random sites in the cell genome. This can lead toinsertional mutagenesis through the interruption of host genes orthrough the insertion of viral regulatory sequences that can interferewith the function of flanking genes (Varmus et al., 1981). Anotherconcern with the use of defective retrovirus vectors is the potentialappearance of wild-type replication-competent virus in the packagingcells. This can result from recombination events in which theintact-sequence from the recombinant virus inserts upstream from thegag, pol, env sequence integrated in the host cell genome. However, newpackaging cell lines are now available that should greatly decrease thelikelihood of recombination (Markowitz et al., 1988; Hersdorffer et al.,1990).

Other viral vectors may be employed as expression constructs in thepresent invention. Vectors derived from viruses such as vaccinia virus(Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988)adeno-associated virus (AAV) (Ridgeway, 1988; Baichwal and Sugden, 1986;Hermonat and Muzycska, 1984) and herpesviruses may be employed. Theyoffer several attractive features for various mammalian cells(Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar etal. 1988; Horwich et al., 1990).

With the recent recognition of defective hepatitis B viruses, newinsight was gained into the structure-function relationship of differentviral sequences. In vitro studies showed that the virus could retain theability for helper-dependent packaging and reverse transcription despitethe deletion of up to 80% of its genome (Horwich et al. 1990). Thissuggested that large portions of the genome could be replaced withforeign genetic material. The hepatotropism and persistence(integration) were particularly attractive properties for liver-directedgene transfer. Chang et al., recently introduced the chloramphenicolacetyltransferase (CAT) gene into duck hepatitis B virus genome in theplace of the polymerase, surface, and pre-surface coding sequences. Itwas co-transfected with wild-type virus into an avian hepatoma cellline. Culture media containing high titers of the recombinant virus wereused to infect primary duckling hepatocytes. Stable CAT gene expressionwas detected for at least 24 days after transfection (Chang et al.,1991).

In order to effect expression of sense or antisense gene constructs, theexpression construct must be delivered into a cell. This delivery may beaccomplished in vitro, as in laboratory procedures for transformingcells lines, or in vivo or ex vivo, as in the treatment of certaindisease states. One mechanism for delivery is via viral infection wherethe expression construct is encapsidated in an infectious viralparticle.

Several non-viral methods for the transfer of expression constructs intocultured mammalian cells also are contemplated by the present invention.These include calcium phosphate precipitation (Graham and Van Der Eb,1973; Chen and Okayama, 1987; Rippe et al., 1990) DEAE-dextran (Gopal,1985), electroporation (Tur-Kaspa et al., 1986; Potter et al., 1984),direct microinjection (Harland and Weintraub, 1985), DNA-loadedliposomes (Nicolau and Sene, 1982; Fraley et al., 1979) andlipofectamine-DNA complexes, cell sonication (Fechheimer et al. 1987),gene bombardment using high velocity microprojectiles (Yang et al.,1990), and receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu,1988). Some of these techniques may be successfully adapted for in vivoor ex vivo use.

Once the expression construct has been delivered into the cell thenucleic acid encoding the gene of interest may be positioned andexpressed at different sites. In certain embodiments, the nucleic acidencoding the gene may be stably integrated into the genome of the cell.This integration may be in the cognate location and orientation viahomologous recombination (gene replacement) or it may be integrated in arandom, non-specific location (gene augmentation). In yet furtherembodiments, the nucleic acid may be stably maintained in the cell as aseparate, episomal segment of DNA. Such nucleic acid segments or“episomes” encode sequences sufficient to permit maintenance andreplication independent of or in synchronization with the host cellcycle. How the expression construct is delivered to a cell and where inthe cell the nucleic acid remains is dependent on the type of expressionconstruct employed.

In yet another embodiment of the invention, the expression construct maysimply consist of naked recombinant DNA or plasmids. Transfer of theconstruct may be performed by any of the methods mentioned above whichphysically or chemically permeabilize the cell membrane. This isparticularly applicable for transfer in vitro but it may be applied toin vivo use as well. Dubensky et al. (1984) successfully injectedpolyomavirus DNA in the form of calcium phosphate precipitates intoliver and spleen of adult and newborn mice demonstrating active viralreplication and acute infection. Benvenisty and Neshif (1986) alsodemonstrated that direct intraperitoneal injection of calciumphosphate-precipitated plasmids results in expression of the transfectedgenes. It is envisioned that DNA encoding a gene of interest may also betransferred in a similar manner in vivo and express the gene product.

In still another embodiment of the invention for transferring a nakedDNA expression construct into cells may involve particle bombardment.This method depends on the ability to accelerate DNA-coatedmicroprojectiles to a high velocity allowing them to pierce cellmembranes and enter cells without killing them (Klein et al., 1987).Several devices for accelerating small particles have been developed.One such device relies on a high voltage discharge to generate anelectrical current, which in turn provides the motive force (Yang etal., 1990). The microprojectiles used have consisted of biologicallyinert substances such as tungsten or gold beads.

Selected organs including the liver, skin, and muscle tissue of rats andmice have been bombarded in vivo (Yang et al. 1990; Zelenin et al.,1991). This may require surgical exposure of the tissue or cells, toeliminate any intervening tissue between the gun and the target organ,i.e., ex vivo treatment. Again, DNA encoding a particular gene may bedelivered via this method and still be incorporated by the presentinvention.

In a further embodiment of the invention, the expression construct maybe entrapped in a liposome. Liposomes are vesicular structurescharacterized by a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh andBachhawat, 1991). Also contemplated are lipofectamine-DNA complexes.

Liposome-mediated nucleic acid delivery and expression of foreign DNA invitro has been very successful. Wong et al., (1980) demonstrated thefeasibility of liposome-mediated delivery and expression of foreign DNAin cultured chick embryo, HeLa and hepatoma cells. Nicolau et al. (1987)accomplished successful liposome-mediated gene transfer in rats afterintravenous injection.

In certain embodiments of the invention, the liposome may be complexedwith a hemagglutinating virus (HVJ). This has been shown to facilitatefusion with the cell membrane and promote cell entry ofliposome-encapsulated DNA (Kaneda et al., 1989). In other embodiments,the liposome may be complexed or employed in conjunction with nuclearnon-histone chromosomal proteins (HfG-1) (Kato et al., 1991). In yetfurther embodiments, the liposome may be complexed or employed inconjunction with both HVJ and HMG-1. In that such expression constructshave been successfully employed in transfer and expression of nucleicacid in vitro and in vivo, then they are applicable for the presentinvention. Where a bacterial promoter is employed in the DNA construct,it also will be desirable to include within the liposome an appropriatebacterial polymerase.

Other expression constructs which can be employed to deliver a nucleicacid encoding a particular gene into cells are receptor-mediateddelivery vehicles. These take advantage of the selective uptake ofmacromolecules by receptor-mediated endocytosis in almost all eukaryoticcells. Because of the cell type-specific distribution of variousreceptors, the delivery can be highly specific (Wu and Wu, 1993).

Receptor-mediated gene targeting vehicles generally consist of twocomponents: a cell receptor-specific ligand and a DNA-binding agent.Several ligands have been used for receptor-mediated gene transfer. Themost extensively characterized ligands are asialoorosomucoid (ASOR) (Wuand Wu, 1987) and transferrin (Wagner et al. 1990). Recently, asynthetic neoglycoprotein, which recognizes the same receptor as ASOR,has been used as a gene delivery vehicle (Ferkol et al., 1993; Peraleset al. 1994) and epidermal growth factor (EGF) has also been used todeliver genes to squamous carcinoma cells (Myers, EPO 0273085).

In other embodiments, the delivery vehicle may comprise a ligand and aliposome. For example, Nicolau et al. (1987) employed lactosyl-ceramide,a galactose-terminal asialganglioside, incorporated into liposomes andobserved an increase in the uptake of the insulin gene by hepatocytes.Thus, it is feasible that a nucleic acid encoding a particular gene alsomay be specifically delivered into a cell type by any number ofreceptor-ligand systems with or without liposomes. For example,epidermal growth factor (EGF) may be used as the receptor for mediateddelivery of a nucleic acid into cells that exhibit upregulation of EGFreceptor. Mannose can be used to target the mannose receptor on livercells Also, antibodies to CD5 (CLL), CD22 (lymphoma), CD25 (T-cellleukemia) and MAA (melanoma) can similarly be used as targetingmoieties.

In certain embodiments, gene transfer may more easily be performed underex vivo conditions. Ex vivo gene therapy refers to the isolation ofcells from an animal, the delivery of a nucleic acid into the cells invitro, and then the return of the modified cells back into an animal.This may involve the surgical removal of tissue/organs from an animal orthe primary culture of cells and tissues.

IV. Generating Antibodies Reactive with MURFs

In another aspect, the present invention contemplates an antibody thatis immunoreactive with a MURF molecule of the present invention, or anyportion thereof An antibody can be a polyclonal or a monoclonalantibody. In a preferred embodiment, an antibody is a monoclonalantibody. Means for preparing and characterizing antibodies are wellknown in the art (see, e.g., Harlow and Lane, 1988).

Briefly, a polyclonal antibody is prepared by immunizing an animal withan immunogen comprising a polypeptide of the present invention andcollecting antisera from that immunized animal. A wide range of animalspecies can be used for the production of antisera. Typically an animalused for production of anti-antisera is a non-human animal includingrabbits, mice, rats, hamsters, pigs or horses. Because of the relativelylarge blood volume of rabbits, a rabbit is a preferred choice forproduction of polyclonal antibodies.

Antibodies, both polyclonal and monoclonal, specific for isoforms ofantigen may be prepared using conventional immunization techniques, aswill be generally known to those of skill in the art. A compositioncontaining antigenic epitopes of the compounds of the present inventioncan be used to immunize one or more experimental animals, such as arabbit or mouse, which will then proceed to produce specific antibodiesagainst the compounds of the present invention. Polyclonal antisera maybe obtained, after allowing time for antibody generation, simply bybleeding the animal and preparing serum samples from the whole blood.

It is proposed that the monoclonal antibodies of the present inventionwill find useful application in standard immunochemical procedures, suchas ELISA and Western blot methods and in immunohistochemical proceduressuch as tissue staining, as well as in other procedures which mayutilize antibodies specific to MURF-related antigen epitopes.Additionally, it is proposed that monoclonal antibodies specific to theparticular MURF of different species may be utilized in other usefulapplications

In general, both polyclonal and monoclonal antibodies against MURFs maybe used in a variety of embodiments. For example, they may be employedin antibody cloning protocols to obtain cDNAs or genes encoding otherMURFs. They may also be used in inhibition studies to analyze theeffects of MURFs related peptides in cells or animals. MURF antibodieswill also be useful in immunolocalization studies to analyze thedistribution of MURFs during various cellular events, for example, todetermine the cellular or tissue-specific distribution of MURFpolypeptides under different points in the cell cycle. A particularlyuseful application of such antibodies is in purifying native orrecombinant MURFs, for example, using an antibody affinity column. Theoperation of all such immunological techniques will be known to those ofskill in the art in light of the present disclosure.

Means for preparing and characterizing antibodies are well known in theart (see, e.g., Harlow and Lane, 1988; incorporated herein byreference). More specific examples of monoclonal antibody preparationare given in the examples below.

As is well known in the art, a given composition may vary in itsimmunogenicity. It is often necessary therefore to boost the host immunesystem, as may be achieved by coupling a peptide or polypeptideimmunogen to a carrier. Exemplary and preferred carriers are keyholelimpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albuminssuch as ovalbumin, mouse serum albumin or rabbit serum albumin can alsobe used as carriers. Means for conjugating a polypeptide to a carrierprotein are well known in the art and include glutaraldehyde,m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide andbis-biazotized benzidine.

As also is well known in the art, the immunogenicity of a particularimmunogen composition can be enhanced by the use of non-specificstimulators of the immune response, known as adjuvants. Exemplary andpreferred adjuvants include complete Freund's adjuvant (a non-specificstimulator of the immune response containing killed Mycobacteriumtuberculosis), incomplete Freund's adjuvants and aluminum hydroxideadjuvant.

The amount of immunogen composition used in the production of polyclonalantibodies varies upon the nature of the immunogen as well as the animalused for immunization. A variety of routes can be used to administer theimmunogen (subcutaneous, intramuscular, intradermal, intravenous andintraperitoneal). The production of polyclonal antibodies may bemonitored by sampling blood of the immunized animal at various pointsfollowing immunization. A second, booster, injection may also be given.The process of boosting and titering is repeated until a suitable titeris achieved. When a desired level of immunogenicity is obtained, theimmunized animal can be bled and the serum isolated and stored, and/orthe animal can be used to generate mAbs.

MAbs may be readily prepared through use of well-known techniques, suchas those exemplified in U.S. Pat. No. 4,196,265, incorporated herein byreference. Typically, this technique involves immunizing a suitableanimal with a selected immunogen composition, e.g., a purified orpartially purified MURF protein, polypeptide or peptide or cellexpressing high levels of MURF. The immunizing composition isadministered in a manner effective to stimulate antibody producingcells. Rodents such as mice and rats are preferred animals, however, theuse of rabbit, sheep frog cells is also possible. The use of rats mayprovide certain advantages (Goding, 1986), but mice are preferred, withthe BALB/c mouse being most preferred as this is most routinely used andgenerally gives a higher percentage of stable fusions.

Following immunization, somatic cells with the potential for producingantibodies, specifically B-lymphocytes (B-cells), are selected for usein the mAb generating protocol. These cells may be obtained frombiopsied spleens, tonsils or lymph nodes, or from a peripheral bloodsample. Spleen cells and peripheral blood cells are preferred, theformer because they are a rich source of antibody-producing cells thatare in the dividing plasmablast stage, and the latter because peripheralblood is easily accessible. Often, a panel of animals will have beenimmunized and the spleen of animal with the highest antibody titer willbe removed and the spleen lymphocytes obtained by homogenizing thespleen with a syringe. Typically, a spleen from an immunized mousecontains approximately 5×10⁷ to 2×10⁸ lymphocytes.

The antibody-producing B lymphocytes from the immunized animal are thenfused with cells of an immortal myeloma cell, generally one of the samespecies as the animal that was immunized. Myeloma cell lines suited foruse in hybridoma-producing fusion procedures preferably arenon-antibody-producing, have high fusion efficiency, and enzymedeficiencies that render then incapable of growing in certain selectivemedia which support the growth of only the desired fused cells(hybridomas).

Any one of a number of myeloma cells may be used, as are known to thoseof skill in the art (Goding, 1986; Campbell, 1984). For example, wherethe immunized animal is a mouse, one may use P3-X63/Ag8, P3-X63-Ag8.653,NS1/1.Ag 4 1, Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 andS194/5XX0 Bul; for rats, one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all usefulin connection with cell fusions.

Methods for generating hybrids of antibody-producing spleen or lymphnode cells and myeloma cells usually comprise mixing somatic cells withmyeloma cells in a 2:1 ratio, though the ratio may vary from about 20:1to about 1:1, respectively, in the presence of an agent or agents(chemical or electrical) that promote the fusion of cell membranes.Fusion methods using Sendai virus have been described (Kohler andMilstein, 1975; 1976), and those using polyethylene glycol (PEG), suchas 37% (v/v) PEG, by Gefter et al., (1977). The use of electricallyinduced fusion methods is also appropriate (Goding, 1986).

Fusion procedures usually produce viable hybrids at low frequencies,around 1×10⁻⁶ to 1×10⁻⁸. However, this does not pose a problem, as theviable, fused hybrids are differentiated from the parental, unfusedcells (particularly the unfused myeloma cells that would normallycontinue to divide indefinitely) by culturing in a selective medium. Theselective medium is generally one that contains an agent that blocks thede novo synthesis of nucleotides in the tissue culture media. Exemplaryand preferred agents are aminopterin, methotrexate, and azaserine.Aminopterin and methotrexate block de novo synthesis of both purines andpyrimidines, whereas azaserine blocks only purine synthesis. Whereaminopterin or methotrexate is used, the media is supplemented withhypoxanthine and thymidine as a source of nucleotides (HAT medium).Where azaserine is used, the media is supplemented with hypoxanthine.

The preferred selection medium is HAT. Only cells capable of operatingnucleotide salvage pathways are able to survive in HAT medium. Themyeloma cells are defective in key enzymes of the salvage pathway, e.g.,hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive.The B-cells can operate this pathway, but they have a limited life spanin culture and generally die within about two weeks. Therefore, the onlycells that can survive in the selective media are those hybrids formedfrom myeloma and B-cells.

This culturing provides a population of hybridomas from which specifichybridomas are selected. Typically, selection of hybridomas is performedby culturing the cells by single-clone dilution in microtiter plates,followed by testing the individual clonal supernatants (after about twoto three weeks) for the desired reactivity. The assay should besensitive, simple and rapid, such as radioimmunoassays, enzymeimmunoassays, cytotoxicity assays, plaque assays, dot immunobindingassays, and the like.

The selected hybridomas would then be serially diluted and cloned intoindividual antibody-producing cell lines, which clones can then bepropagated indefinitely to provide mAbs. The cell lines may be exploitedfor mAb production in two basic ways. A sample of the hybridoma can beinjected (often into the peritoneal cavity) into a histocompatibleanimal of the type that was used to provide the somatic and myelomacells for the original fusion. The injected animal develops tumorssecreting the specific monoclonal antibody produced by the fused cellhybrid. The body fluids of the animal, such as serum or ascites fluid,can then be tapped to provide mAbs in high concentration. The individualcell lines could also be cultured in vitro, where the mAbs are naturallysecreted into the culture medium from which they can be readily obtainedin high concentrations. mAbs produced by either means may be furtherpurified, if desired, using filtration, centrifugation and variouschromatographic methods such as HPLC or affinity chromatography.

V. Diagnosing and Treating Defects in MURFs

The inventors have shown MURFs play an important role in thestabilization of microtubules. In addition, in diseased heart tissue,MURF activity is reduced, implicating that MURFs, possibly throughmicrotubule stabilization, play in important role in normal cardiacfunction. Thus, in another embodiment, there are provided methods fordiagnosing defects in MURF expression and function. More specifically,point mutations, deletions, insertions or regulatory pertubationsrelating to MURFs may be assessed using standard technologies, asdescribed below.

A. Genetic Diagnosis

One embodiment of the instant invention comprises a method for detectingvariation in the expression of MURF-1, MURF-2 or MURF-3. This maycomprises determining that level of MURF-1, MURF-2 or MURF-3 ordetermining specific alterations in the expressed product.

A suitable biological sample can be any tissue or fluid. Variousembodiments include cells of the skin, muscle, facia, brain, prostate,breast, endometrium, lung, head & neck, pancreas, small intestine, bloodcells, liver, testes, ovaries, colon, skin, stomach, esophagus, spleen,lymph node, bone marrow or kidney. Other embodiments include fluidsamples such as peripheral blood, lymph fluid, ascites, serous fluid,pleural effusion, sputum, cerebrospinal fluid, lacrimal fluid, stool orurine.

Nucleic acid used is isolated from cells contained in the biologicalsample, according to standard methodologies (Sambrook et al., 1989). Thenucleic acid may be genomic DNA or fractionated or whole cell RNA. WhereRNA is used, it may be desired to convert the RNA to a complementaryDNA. In one embodiment, the RNA is whole cell RNA; in another, it ispoly-A RNA. Normally, the nucleic acid is amplified.

Depending on the format, the specific nucleic acid of interest isidentified in the sample directly using amplification or with a second,known nucleic acid following amplification. Next, the identified productis detected. In certain applications, the detection may be performed byvisual means (e.g. ethidium bromide staining of a gel). Alternatively,the detection may involve indirect identification of the product viachemiluminescence, radioactive scintigraphy of radiolabel or fluorescentlabel or even via a system using electrical or thermal impulse signals(Affymax Technology, Bellus, 1994).

Various types of defects may be identified by the present methods. Thus,“alterations” should be read as including deletions, insertions, pointmutations and duplications. Point mutations result in stop codons,frameshift mutations or amino acid substitutions. Somatic mutations arethose occurring in non-germline tissues. Germ-line tissue can occur inany tissue and are inherited. Mutations in and outside the coding regionalso may affect the amount of MURF produced, both by altering thetranscription of the gene or in destabilizing or otherwise altering theprocessing of either the transcript (mRNA) or protein.

It is contemplated that other mutations in the MURF genes may beidentified in accordance with the present inevntion. A variety ofdifferent assays are contemplated in this regard, including but notlimited to, fluorescent in situ hybridization (FISH), direct DNAsequencing, PFGE analysis, Southern or Northern blotting,single-stranded conformation analysis (SSCA), RNAse protection assay,allele-specific oligonucleotide (ASO), dot blot analysis, denaturinggradient gel electrophoresis, RFLP and PCR™-SSCP.

(i) Primers and Probes

The term primer, as defined herein, is meant to encompass any nucleicacid that is capable of priming the synthesis of a nascent nucleic acidin a template-dependent process. Typically, primers are oligonucleotidesfrom ten to twenty base pairs in length, but longer sequences can beemployed. Primers may be provided in double-stranded or single-strandedform, although the single-stranded form is preferred. Probes are defineddifferently, although they may act as primers. Probes, while perhapscapable of priming, are designed to binding to the target DNA or RNA andneed not be used in an amplification process.

In preferred embodiments, the probes or primers are labeled withradioactive species (³²P, ¹⁴C, ³⁵S, ³H, or other label), with afluorophore (rhodamine, fluorescein) or a chemillumiscent (luciferase).

(ii) Template Dependent Amplification Methods

A number of template dependent processes are available to amplify themarker sequences present in a given template sample. One of the bestknown amplification methods is the polymerase chain reaction (referredto as PCR™) which is described in detail in U.S. Pat. Nos. 4,683,195,4,683,202 and 4,800,159, and in Innis et al., 1990, each of which isincorporated herein by reference in its entirety.

Briefly, in PCR™, two primer sequences are prepared that arecomplementary to regions on opposite complementary strands of the markersequence. An excess of deoxynucleoside triphosphates are added to areaction mixture along with a DNA polymerase, e.g., Taq polymerase. Ifthe marker sequence is present in a sample, the primers will bind to themarker and the polymerase will cause the primers to be extended alongthe marker sequence by adding on nucleotides. By raising and loweringthe temperature of the reaction mixture, the extended primers willdissociate from the marker to form reaction products, excess primerswill bind to the marker and to the reaction products and the process isrepeated.

A reverse transcriptase PCR™ amplification procedure may be performed inorder to quantify the amount of mRNA amplified. Methods of reversetranscribing RNA into cDNA are well known and described in Sambrook etal. 1989. Alternative methods for reverse transcription utilizethermostable, RNA-dependent DNA polymerases. These methods are describedin WO 90/07641 filed Dec. 21, 1990. Polymerase chain reactionmethodologies are well known in the art.

Another method for amplification is the ligase chain reaction (“LCR”),disclosed in EPO No. 320 308, incorporated herein by reference in itsentirety. In LCR, two complementary probe pairs are prepared, and in thepresence of the target sequence, each pair will bind to oppositecomplementary strands of the target such that they abut. In the presenceof a ligase, the two probe pairs will link to form a single unit. Bytemperature cycling, as in PCR™, bound ligated units dissociate from thetarget and then serve as “target sequences” for ligation of excess probepairs. U.S. Pat. No. 4,883,750 describes a method similar to LCR forbinding probe pairs to a target sequence.

Methods based on ligation of two (or more) oligonucleotides in thepresence of nucleic acid having the sequence of the resulting“di-oligonucleotide”, thereby amplifying the di-oligonucleotide, mayalso be used in the amplification step of the present invention. Wu etal. (1989), incorporated herein by reference in its entirety.

(iii) Southern/Northern Blotting

Blotting techniques are well known to those of skill in the art.Southern blotting involves the use of DNA as a target, whereas Northernblotting involves the use of RNA as a target. Each provide differenttypes of information, although cDNA blotting is analogous, in manyaspects, to blotting or RNA species.

Briefly, a probe is used to target a DNA or RNA species that has beenimmobilized on a suitable matrix, often a filter of nitrocellulose. Thedifferent species should be spatially separated to facilitate analysis.This often is accomplished by gel electrophoresis of nucleic acidspecies followed by “blotting” on to the filter.

Subsequently, the blotted target is incubated with a probe (usuallylabeled) under conditions that promote denaturation and rehybridization.Because the probe is designed to base pair with the target, the probewill binding a portion of the target sequence under renaturingconditions. Unbound probe is then removed, and detection is accomplishedas described above.

(iv) Separation Methods

It normally is desirable, at one stage or another, to separate theamplification product from the template and the excess primer for thepurpose of determining whether specific amplification has occurred. Inone embodiment, amplification products are separated by agarose,agarose-acrylamide or polyacrylamide gel electrophoresis using standardmethods. See Sambrook et al. 1989.

Alternatively, chromatographic techniques may be employed to effectseparation. There are many kinds of chromatography which may be used inthe present invention: adsorption, partition, ion-exchange and molecularsieve, and many specialized techniques for using them including column,paper, thin-layer and gas chromatography (Freifelder, 1982).

(v) Detection Methods

Products may be visualized in order to confirm amplification of themarker sequences. One typical visualization method involves staining ofa gel with ethidium bromide and visualization under UV light.Alternatively, if the amplification products are integrally labeled withradio- or fluorometrically-labeled nucleotides, the amplificationproducts can then be exposed to x-ray film or visualized under theappropriate stimulating spectra, following separation.

In one embodiment, visualization is achieved indirectly. Followingseparation of amplification products, a labeled nucleic acid probe isbrought into contact with the amplified marker sequence. The probepreferably is conjugated to a chromophore but may be radiolabeled. Inanother embodiment, the probe is conjugated to a binding partner, suchas an antibody or biotin, and the other member of the binding paircarries a detectable moiety.

In one embodiment, detection is by a labeled probe. The techniquesinvolved are well known to those of skill in the art and can be found inmany standard books on molecular protocols. See Sambrook et al., 1989.For example, chromophore or radiolabel probes or primers identify thetarget during or following amplification.

One example of the foregoing is described in U.S. Pat. No. 5,279,721,incorporated by reference herein, which discloses an apparatus andmethod for the automated electrophoresis and transfer of nucleic acids.The apparatus permits electrophoresis and blotting without externalmanipulation of the gel and is ideally suited to carrying out methodsaccording to the present invention.

In addition, the amplification products described above may be subjectedto sequence analysis to identify specific kinds of variations usingstandard sequence analysis techniques. Within certain methods,exhaustive analysis of genes is carried out by sequence analysis usingprimer sets designed for optimal sequencing (Pignon et al, 1994). Thepresent invention provides methods by which any or all of these types ofanalyses may be used. Using the sequences disclosed herein,oligonucleotide primers may be designed to permit the amplification ofsequences throughout the MURF genes that may then be analyzed by directsequencing.

(vi) Kit Components

All the essential materials and reagents required for detecting andsequencing MURF-1, MURF-2 and MURF-3 and variants thereof may beassembled together in a kit. This generally will comprise preselectedprimers and probes. Also included may be enzymes suitable for amplifyingnucleic acids including various polymerases (RT, Taq, Sequenase™ etc.),deoxynucleotides and buffers to provide the necessary reaction mixturefor amplification. Such kits also generally will comprise, in suitablemeans, distinct containers for each individual reagent and enzyme aswell as for each primer or probe.

B. Immunologic Diagnosis

Antibodies of the present invention can be used in characterizing theMURF content of healthy and diseased tissues, through techniques such asELISAs and Western blotting. This may provide a screen for the presenceor absence of cardiomyopathy or as a predictor of heart disease.

The use of antibodies of the present invention, in an ELISA assay iscontemplated. For example, anti-MURF antibodies are immobilized onto aselected surface, preferably a surface exhibiting a protein affinitysuch as the wells of a polystyrene microtiter plate. After washing toremove incompletely adsorbed material, it is desirable to bind or coatthe assay plate wells with a non-specific protein that is known to beantigenically neutral with regard to the test antisera such as bovineserum albumin (BSA), casein or solutions of powdered milk. This allowsfor blocking of non-specific adsorption sites on the immobilizingsurface and thus reduces the background caused by non-specific bindingof antigen onto the surface.

After binding of antibody to the well, coating with a non-reactivematerial to reduce background, and washing to remove unbound material,the immobilizing surface is contacted with the sample to be tested in amanner conducive to immune complex (antigen/antibody) formation.

Following formation of specific immunocomplexes between the test sampleand the bound antibody, and subsequent washing, the occurrence and evenamount of immunocomplex formation may be determined by subjecting sameto a second antibody having specificity for MURF-1, MURF-2 or MURF-3that differs the first antibody. Appropriate conditions preferablyinclude diluting the sample with diluents such as BSA, bovine gammaglobulin (BGG) and phosphate buffered saline (PBS)/Tween®. These addedagents also tend to assist in the reduction of nonspecific background.The layered antisera is then allowed to incubate for from about 2 toabout 4 hr, at temperatures preferably on the order of about 25° toabout 27° C. Following incubation, the antisera-contacted surface iswashed so as to remove non-immunocomplexed material. A preferred washingprocedure includes washing with a solution such as PBS/Tween®, or boratebuffer.

To provide a detecting means, the second antibody will preferably havean associated enzyme that will generate a color development uponincubating with an appropriate chromogenic substrate. Thus, for example,one will desire to contact and incubate the second antibody-boundsurface with a urease or peroxidase-conjugated anti-human IgG for aperiod of time and under conditions which favor the development ofimmunocomplex formation (e.g., incubation for 2 hr at room temperaturein a PBS-containing solution such as PBS/Tween®).

After incubation with the second enzyme-tagged antibody, and subsequentto washing to remove unbound material, the amount of label is quantifiedby incubation with a chromogenic substrate such as urea and bromocresolpurple or 2,2′-azino-di-(3-ethyl-benzthiazoline)-6-sulfonic acid (ABTS)and H₂O₂, in the case of peroxidase as the enzyme label. Quantitation isthen achieved by measuring the degree of color generation, e.g., using avisible spectrum spectrophotometer.

The preceding format may be altered by first binding the sample to theassay plate. Then, primary antibody is incubated with the assay plate,followed by detecting of bound primary antibody using a labeled secondantibody with specificity for the primary antibody.

The antibody compositions of the present invention will find great usein immunoblot or Western blot analysis. The antibodies may be used ashigh-affinity primary reagents for the identification of proteinsimmobilized onto a solid support matrix, such as nitrocellulose, nylonor combinations thereof In conjunction with immunoprecipitation,followed by gel electrophoresis, these may be used as a single stepreagent for use in detecting antigens against which secondary reagentsused in the detection of the antigen cause an adverse background.Immunologically-based detection methods for use in conjunction withWestern blotting include enzymatically-, radiolabel-, orfluorescently-tagged secondary antibodies against the toxin moiety areconsidered to be of particular use in this regard.

C. Treating Defects in MURF Expression or Function

The present invention also involves, in another embodiment, thetreatment of disease states related to the aberrant expression and/orfunction of MURFs. In particular, it is envisioned that reduced MURFactivity plays a role in microtubule destabilization and cardiacfailure. Thus, increasing levels of MURFs, or compensating for mutationsthat reduce or eliminate the activity of MURFs, are believed to providetherapeutic intervention in cardiomyopathies.

In addition, by increasing levels of MURFs, it is possible that defectsin other cardiac genes may be compensated for. As discussed above, MURFsbind to and stabilize microtubules. Thus, increasing the expression ofMURFs can stabilize the cytoskeleton of failing heart. Similarly, insituations where increase heart muscle mass is desired, inhibiting MURFexpression could stimulate proliferation.

D. Genetic Based Therapies

One of the therapeutic embodiments contemplated by the present inventorsis the intervention, at the molecular level, in the events involved incardiac failure. Specifically, the present inventors intend to provide,to a cardiac cell, an expression construct capable of providing MURF-1,MURF-2 or MURF-3 to that cell. Because the sequence homology between thehuman, mouse and Drosophila genes, any of these nucleic acids could beused in human therapy, as could any of the gene sequence variantsdiscussed above which would encode the same, or a biologicallyequivalent polypeptide. The lengthy discussion of expression vectors andthe genetic elements employed therein is incorporated into this sectionby reference. Particularly preferred expression vectors are viralvectors such as adenovirus, adeno-associated virus, herpesvirus,vaccinia virus and retrovirus. Also preferred isliposomally-encapsulated expression vector.

Those of skill in the art are well aware of how to apply gene deliveryto in vivo situations. For viral vectors, one generally will prepare aviral vector stock. Depending on the kind of virus and the titerattainable, one will deliver 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹,1×10¹⁰, 1×10¹¹ or 1×10¹² infectious particles to the patient. Similarfigures may be extrapolated for liposomal or other non-viralformulations by comparing relative uptake efficiencies. Formulation as apharmaceutically acceptable composition is discussed below. Variousroutes are contemplated, but local provision to the heart and systemicprovision (intraarterial or intravenous) are preferred.

E. Combined Therapy

In many clinical situations, it is advisable to use a combination ofdistinct therapies. Thus, it is envisioned that, in addition to thetherapies described above, one would also wish to provide to the patientmore “standard” pharmaceutical cardiac therapies. Examples of standardtherapies include so-called “beta blockers”, anti-hypertensives,cardiotonics, anti-thrombotics, vasodilators, hormone antagonists,endothelin antagonists, cytokine inhibitors/blockers, calcium channelblockers, phosphodiesterase inhibitors and angiotensin type 2antagonists. Also envisioned are combinations with pharmaceuticalsidentified according to the screening methods described herein.

Combinations may be achieved by contacting cardiac cells with a singlecomposition or pharmacological formulation that includes both agents, orby contacting the cell with two distinct compositions or formulations,at the same time, wherein one composition includes the expressionconstruct and the other includes the agent. Alternatively, gene therapymay precede or follow the other agent treatment by intervals rangingfrom minutes to weeks. In embodiments where the other agent andexpression construct are applied separately to the cell, one wouldgenerally ensure that a significant period of time did not expirebetween the time of each delivery, such that the agent and expressionconstruct would still be able to exert an advantageously combined effecton the cell. In such instances, it is contemplated that one wouldcontact the cell with both modalities within about 12-24 hours of eachother and, more preferably, within about 6-12 hours of each other, witha delay time of only about 12 hours being most preferred. In somesituations, it may be desirable to extend the time period for treatmentsignificantly, however, where several days (2, 3, 4, 5, 6 or 7) toseveral weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respectiveadministrations.

It also is conceivable that more than one administration of either aMURF-1, MURF-2 or MURF-3 gene, or the other agent will be desired.Various combinations may be employed, where MURF is “A” and the otheragent is “B”, as exemplified below:

A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B

A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A

A/A/A/B B/A/A/A A/B/A/A A/A/B/A A/B/B/B B/A/B/B B/B/A/B

Other combinations are contemplated as well.

F. Formulations and Routes for Administration to Patients

Where clinical applications are contemplated, it will be necessary toprepare pharmaceutical compositions—expression vectors, virus stocks anddrugs—in a form appropriate for the intended application. Generally,this will entail preparing compositions that are essentially free ofpyrogens, as well as other impurities that could be harmful to humans oranimals.

One will generally desire to employ appropriate salts and buffers torender delivery vectors stable and allow for uptake by target cells.Buffers also will be employed when recombinant cells are introduced intoa patient. Aqueous compositions of the present invention comprise aneffective amount of the vector to cells, dissolved or dispersed in apharmaceutically acceptable carrier or aqueous medium. Such compositionsalso are referred to as inocula. The phrase “pharmaceutically orpharmacologically acceptable” refer to molecular entities andcompositions that do not produce adverse, allergic, or other untowardreactions when administered to an animal or a human As used herein,“pharmaceutically acceptable carrier” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents and the like. The use of suchmedia and agents for pharmaceutically active substances is well know inthe art. Except insofar as any conventional media or agent isincompatible with the vectors or cells of the present invention, its usein therapeutic compositions is contemplated. Supplementary activeingredients also can be incorporated into the compositions.

The active compositions of the present invention may include classicpharmaceutical preparations. Administration of these compositionsaccording to the present invention will be via any common route so longas the target tissue is available via that route. This includes oral,nasal, buccal, rectal, vaginal or topical. Alternatively, administrationmay be by orthotopic, intradermal, subcutaneous, intramuscular,intraperitoneal or intravenous injection. Such compositions wouldnormally be administered as pharmaceutically acceptable compositions,described supra.

The active compounds may also be administered parenterally orintraperitoneally. Solutions of the active compounds as free base orpharmacologically acceptable salts can be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose. Dispersions canalso be prepared in glycerol, liquid polyethylene glycols, and mixturesthereof and in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), suitable mixtures thereof,and vegetable oils. The proper fluidity can be maintained, for example,by the use of a coating, such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial an antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutical active substances is well knownin the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

For oral administration the polypeptides of the present invention may beincorporated with excipients and used in the form of non-ingestiblemouthwashes and dentifrices. A mouthwash may be prepared incorporatingthe active ingredient in the required amount in an appropriate solvent,such as a sodium borate solution (Dobell's Solution). Alternatively, theactive ingredient may be incorporated into an antiseptic wash containingsodium borate, glycerin and potassium bicarbonate. The active ingredientmay also be dispersed in dentifrices, including: gels, pastes, powdersand slurries. The active ingredient may be added in a therapeuticallyeffective amount to a paste dentifrice that may include water, binders,abrasives, flavoring agents, foaming agents, and humectants.

The compositions of the present invention may be formulated in a neutralor salt form. Pharmaceutically-acceptable salts include the acidaddition salts (formed with the free amino groups of the protein) andwhich are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms such as injectable solutions, drug release capsules and thelike. For parenteral administration in an aqueous solution, for example,the solution should be suitably buffered if necessary and the liquiddiluent first rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, sterile aqueous media which can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage could be dissolved in I ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. The person responsible for administration will,in any event, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, general safety and purity standards as required by FDAOffice of Biologics standards.

VI. Methods of Making Transgenic Mice

A particular embodiment of the present invention provides transgenicanimals that contain MURF-related constructs. Transgenic animalsexpressing MURF-1 and MURF-2, recombinant cell lines derived from suchanimals, and transgenic embryos may be useful in methods for screeningfor and identifying agents that modulate the function of MURF-1 orMURF-2, and thereby alleviate pathology related to the over or underexpression of these molecules. The use of constitutively expressed MURFsprovides a model for over- or unregulated expression. Also, transgenicanimals which are “knocked out” for MURF-1 and/or MURF-2 will find usein analysis of developmental aspects of MURFs.

A. Methods of Producing Transgenics

In a general aspect, a transgenic animal is produced by the integrationof a given transgene into the genome in a manner that permits theexpression of the transgene. Methods for producing transgenic animalsare generally described by Wagner and Hoppe (U.S. Pat. No. 4,873,191;which is incorporated herein by reference), Brinster et al. 1985; whichis incorporated herein by reference in its entirety) and in“Manipulating the Mouse Embryo; A Laboratory Manual” 2nd edition (eds.,Hogan, Beddington, Costantimi and Long, Cold Spring Harbor LaboratoryPress, 1994; which is incorporated herein by reference in its entirety).

Typically, a gene flanked by genomic sequences is transferred bymicroinjection into a fertilized egg. The microinjected eggs areimplanted into a host female, and the progeny are screened for theexpression of the transgene. Transgenic animals may be produced from thefertilized eggs from a number of animals including, but not limited toreptiles, amphibians, birds, mammals, and fish.

DNA clones for microinjection can be prepared by any means known in theart. For example, DNA clones for microinjection can be cleaved withenzymes appropriate for removing the bacterial plasmid sequences, andthe DNA fragments electrophoresed on 1% agarose gels in TBE buffer,using standard techniques. The DNA bands are visualized by staining withethidium bromide, and the band containing the expression sequences isexcised. The excised band is then placed in dialysis bags containing 0.3M sodium acetate, pH 7.0. DNA is electroeluted into the dialysis bags,extracted with a 1:1 phenol:chloroform solution and precipitated by twovolumes of ethanol. The DNA is redissolved in 1 ml of low salt buffer(0.2 M NaCl, 20 mM Tris,pH 7.4, and 1 mM EDTA) and purified on anElutip-D™ column. The column is first primed with 3 ml of high saltbuffer (1 M NaCl, 20 mM Tris, pH 7.4, and 1 mM EDTA) followed by washingwith 5 ml of low salt buffer. The DNA solutions are passed through thecolumn three times to bind DNA to the column matrix. After one wash with3 ml of low salt buffer, the DNA is eluted with 0.4 ml high salt bufferand precipitated by two volumes of ethanol. DNA concentrations aremeasured by absorption at 260 nm in a UV spectrophotometer. Formicroinjection, DNA concentrations are adjusted to 3 μg/ml in 5 mM Tris,pH 7.4 and 0.1 mM EDTA.

Other methods for purification of DNA for microinjection are describedin Hogan et al. Manipulating the Mouse Embryo (Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., 1986), in Palmiter et al. Nature300:611 (1982); in The Qiagenologist, Application Protocols, 3rdedition, published by Qiagen, Inc., Chatsworth, Calif.; and in Sambrooket al. Molecular Cloning: A Laboratory Manual (Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., 1989).

In an exemplary microinjection procedure, female mice six weeks of ageare induced to superovulate with a 5 IU injection (0.1 cc, ip) ofpregnant mare serum gonadotropin (PMSG; Sigma) followed 48 hours laterby a 5 IU injection (0.1 cc, ip) of human chorionic gonadotropin (hCG,Sigma). Females are placed with males immediately after hCG injection.Twenty-one hours after hCG injection, the mated females are sacrificedby CO₂ asphyxiation or cervical dislocation and embryos are recoveredfrom excised oviducts and placed in Dulbecco's phosphate buffered salinewith 0.5% bovine serum albumin (BSA, Sigma). Surrounding cumulus cellsare removed with hyaluronidase (1 mg/ml). Pronuclear embryos are thenwashed and placed in Earle's balanced salt solution containing 0.5% BSA(EBSS) in a 37.5° C. incubator with a humidified atmosphere at 5% CO₂,95% air until the time of injection. Embryos can be implanted at thetwo-cell stage.

Randomly cycling adult female mice are paired with vasectomized males.C57BL/6 or Swiss mice or other comparable strains can be used for thispurpose. Recipient females are mated at the same time as donor females.At the time of embryo transfer, the recipient females are anesthetizedwith an intraperitoneal injection of 0.015 ml of 2.5% avertin per gramof body weight. The oviducts are exposed by a single midline dorsalincision. An incision is then made through the body wall directly overthe oviduct. The ovarian bursa is then torn with watchmakers forceps.Embryos to be transferred are placed in DPBS (Dulbecco's phosphatebuffered saline) and in the tip of a transfer pipet (about 10 to 12embryos). The pipet tip is inserted into the infundibulum and theembryos transferred. After the transfer, the incision is closed by twosutures.

B. Disease Models

Microtubule depolymerization is also required for mitosis. Thus, theability of MURF to prevent microtubule depolymerization is particularlyintriguing in that striated muscle cells are irreversibly postmitotic.In this regard, MURF-transfected cells are unable to divide. Thus, it iscontemplated that MURF trangenics can be useful to explore pathologiesrelated to microtubule depolymerization and cell division. In addition,it may be possible to screen MURF transfected cells for compounds(peptides, combinatorial chemical libraries) with the potential tostimulate DNA synthesis. Such compounds would have utility in promotingcardiomyocyte proliferation and thereby benefit cardiac regeneration inresponse to damage.

Microtubules have been shown to play important roles in regulatingmuscle cell morphology, sarcomere assembly, and function. In skeletalmuscle cells, the microtubule network is reorganized to form stablemicrotubules during differentiation (Gunderson et al., 1989). Integrityof the microtubular array is essential for proper alignment of myoblastsduring fusion and for correct formation of myofibrils (Antin et al.,1981; Toyama et al., 1982).

Consistent with the conclusion that MURF is an integral component of themechanism that regulates microtubule stability in muscle cells, bothskeletal and cardiac muscle have been demonstrated to possess apopulation of microtubules that is stable to depolymerization and coldshock (Webster, 1997). Moreover, expression of MURF mirrors theaccumulation of stable glutamic acid-modified tubulin during theformation of skeletal muscle in vitro. A muscle-specific isoform of MAP4also is expressed upon differentiation in a pattern very similar to thatof MURF (Mangan and Olmsted, 1996). However, inhibition of MAP4expression during differentiation does not block myotube formation butcauses the accumulation of multinucleated myotubes with a roundedmorphology

Microtubule stabilization and increased microtubule density also havebeen proposed as a mechanism for contractile dysfunction in cardiachypertrophy (Sato et al., 1997). There is evidence that stabilization ofthe microtubule array in hypertrophic cardiomyocytes is mediated by amicrotubule-associated protein, that is as yet unidentified (Sato et al.1997). MAP4 expression has been demonstrated to be induced duringpressure overload-induced hypertrophy in cats (Sato et al, 1997). MAP4has been demonstrated to stabilize microtubules in vitro making it aplausable candidate for such a factor (Kaech et al., 1996; Nguyen etal., 1997). Interestingly, inhibiting expression of MAP4 in fibroblaststdoes not adversely affect microtubule dynamics, suggesting a specializedrole for MAP4 in a muscle environment or functional redundancy betweenmembers of the MAP family (Wang et al., 1996). The striated musclerestricted expression pattern and the microtubule stabilizing effects ofMURF make it an interesting candidate for a factor contributing to thecontractile dysfunction in cardiac hypertrophy.

VII. Screening Assays

Several human diseases have been linked to mutations in genes encodingmicrotubule binding proteins, demonstrating the importance of themicrotubule network for normal patterning and development. For example,Mid1, Doublecortin, Lisl, and Tau, all microtubule-associated factorscapable of stabilizing microtubules, have been implicated in theprogression of disease (Gleeson et al., 1999; Kosik, 1990; Koulakoff etal. 1999; Lo Nigro et al., 1997; Quaderi et al., 1997; Tolnay andProbst, 1999). Mutant mice lacking the microtubule-binding proteinsKif3A and Kif3B of the kinesin superfamily, have also been shown todisplay situs inversus of the heart and other organs (Marszalek et al.1999; Takeda et al. 1999). A similar phenotype is observed in miceharboring a deleted ATP binding domain of left-right dynein (Supp etal., 1999).

Thus, the present invention also contemplates the screening of compoundsfor various abilities to interact and/or affect MURF-1, MURF-2 andMURF-3 expression or function. Particularly preferred compounds will bethose useful in inhibiting or promoting the actions of MURF-1, MURF-2and MURF-3 on microtubules (e.g., tubulin) cardiac hypertrophy andpreventing or reversing heart disease. In the screening assays of thepresent invention, the candidate substance may first be screened forbasic biochemical activity—e.g., binding to a target molecule—and thentested for its ability to inhibit modulate expression, at the cellular,tissue or whole animal level.

A. Modulators and Assay Formats

i) Assay Formats

The present invention provides methods of screening for modulators ofMURF-1, MURF-2 and MURF-3 expression and binding to microtubules. In oneembodiment, the present invention is directed to a method of:

(i) providing a MURF-1, MURF-2 or MURF-3 polypeptide;

(ii) contacting the MURF-1, MURF-2 or MURF-3 polypeptide with thecandidate substance; and

(iii) determining the binding of the candidate substance to the MURF-1,MURF-2 or MURF-3 polypeptide.

In another embodiment, this assay can be easily modified to look at thecandidate substances effects on MURF-1, MURF-2 and/or MURF-3 binding tomicrotubules, intermediate filaments or homo- or heterodimerization.

In yet another embodiment, the assay looks not at binding, but at MURFfunction. Such methods would comprise, for example:

(i) providing a cell that expresses MURF-1, MURF-2 or MURF-3polypeptide;

(ii) contacting the cell with the candidate substance; and

(iii) determining the effect of the candidate substance on glutamic acidmodification of microtubules.

In still yet other embodiments, one would look at the effect of acandidate substance on the expression of MURFs. This can be done byexamining mRNA expression, although alterations in mRNA stability andtranslation would not be accounted for. A more direct way of assessingexpression is by directly examining protein levels, for example, throughWestern blot or ELISA.

ii) Inhibitors and Activators

An inhibitor according to the present invention may be one which exertsan inhibitory effect on the expression of function of MURF-1, MURF-2and/or MURF-3. By the same token, an activator according to the presentinvention may be one which exerts a stimulatory effect on the expressionof function of MURF-1, MURF-2 and/or MURF-3.

iii) Candidate Substances

As used herein, the term “candidate substance” refers to any moleculethat may potentially modulate MURF-1, MURF-2 and/or MURF-3 expression orfunction. The candidate substance may be a protein or fragment thereof,a small molecule inhibitor, or even a nucleic acid molecule. It mayprove to be the case that the most useful pharmacological compounds willbe compounds that are structurally related to compounds which interactnaturally with MURF-1, MURF-2 and/or MURF-3. Creating and examining theaction of such molecules is known as “rational drug design,” and includemaking predictions relating to the structure of target molecules.

The goal of rational drug design is to produce structural analogs ofbiologically active polypeptides or target compounds. By creating suchanalogs, it is possible to fashion drugs which are more active or stablethan the natural molecules, which have different susceptibility toalteration or which may affect the function of various other molecules.In one approach, one would generate a three-dimensional structure for amolecule like a MURF, and then design a molecule for its ability tointeract with MURF Alternatively, one could design a partiallyfunctional fragment of a MURF (binding but no activity), therebycreating a competitive inhibitor. This could be accomplished by x-raycrystallography, computer modeling or by a combination of bothapproaches.

It also is possible to use antibodies to ascertain the structure of atarget compound or inhibitor. In principle, this approach yields apharmacore upon which subsequent drug design can be based. It ispossible to bypass protein crystallography altogether by generatinganti-idiotypic antibodies to a functional, pharmacologically activeantibody. As a mirror image of a mirror image, the binding site ofanti-idiotype would be expected to be an analog of the original antigen.The anti-idiotype could then be used to identify and isolate peptidesfrom banks of chemically- or biologically-produced peptides. Selectedpeptides would then serve as the pharmacore. Anti-idiotypes may begenerated using the methods described herein for producing antibodies,using an antibody as the antigen.

On the other hand, one may simply acquire, from various commercialsources, small molecule libraries that are believed to meet the basiccriteria for useful drugs in an effort to “brute force” theidentification of useful compounds. Screening of such libraries,including combinatorially generated libraries (e.g., peptide libraries),is a rapid and efficient way to screen large number of related (andunrelated) compounds for activity. Combinatorial approaches also lendthemselves to rapid evolution of potential drugs by the creation ofsecond, third and fourth generation compounds modeled of active, butotherwise undesirable compounds.

Candidate compounds may include fragments or parts ofnaturally-occurring compounds or may be found as active combinations ofknown compounds which are otherwise inactive. It is proposed thatcompounds isolated from natural sources, such as animals, bacteria,fungi, plant sources, including leaves and bark, and marine samples maybe assayed as candidates for the presence of potentially usefulpharmaceutical agents. It will be understood that the pharmaceuticalagents to be screened could also be derived or synthesized from chemicalcompositions or man-made compounds. Thus, it is understood that thecandidate substance identified by the present invention may bepolypeptide, polynucleotide, small molecule inhibitors or any othercompounds that may be designed through rational drug design startingfrom known inhibitors of hypertrophic response.

Other suitable inhibitors include antisense molecules, ribozymes, andantibodies (including single chain antibodies).

It will, of course, be understood that all the screening methods of thepresent invention are useful in themselves notwithstanding the fact thateffective candidates may not be found. The invention provides methodsfor screening for such candidates, not solely methods of finding them.

B. In vitro Assays

A quick, inexpensive and easy assay to run is a binding assay. Bindingof a molecule to a target may, in and of itself, be inhibitory, due tosteric, allosteric or charge—charge interactions. This can be performedin solution or on a solid phase and can be utilized as a first roundscreen to rapidly eliminate certain compounds before moving into moresophisticated screening assays. In one embodiment of this kind, thescreening of compounds that bind to a MURF-1, MURF-2 or MURF-3 moleculeor fragment thereof is provided.

The target may be either free in solution, fixed to a support, expressedin or on the surface of a cell. Either the target or the compound may belabeled, thereby permitting determining of binding. In anotherembodiment, the assay may measure the inhibition of binding of a targetto a natural or artificial substrate or binding partner (such as aMURF). Competitive binding assays can be performed in which one of theagents (MURF for example) is labeled. Usually, the target will be thelabeled species, decreasing the chance that the labeling will interferewith the binding moiety's function. One may measure the amount of freelabel versus bound label to determine binding or inhibition of binding.

A technique for high throughput screening of compounds is described inWO 84/03564. Large numbers of small peptide test compounds aresynthesized on a solid substrate, such as plastic pins or some othersurface. The peptide test compounds are reacted with, for example, aMURF and washed. Bound polypeptide is detected by various methods.

Purified target, such as a MURF, can be coated directly onto plates foruse in the aforementioned drug screening techniques. However,non-neutralizing antibodies to the polypeptide can be used to immobilizethe polypeptide to a solid phase. Also, fusion proteins containing areactive region (preferably a terminal region) may be used to link anactive region (e.g., the C-terminus of a MURF) to a solid phase.

C. In Cyto Assays

Various cell lines that express MURF-1 MURF-2 and or MURF-2 can beutilized for screening of candidate substances. For example, cellscontaining a MURF with an engineered indicators can be used to studyvarious functional attributes of candidate compounds. In such assays,the compound would be formulated appropriately, given its biochemicalnature, and contacted with a target cell.

Depending on the assay, culture may be required. As discussed above, thecell may then be examined by virtue of a number of different physiologicassays (growth, size, Ca⁺⁺ effects). Alternatively, molecular analysismay be performed in which the function of a MURF and related pathwaysmay be explored. This involves assays such as those for proteinexpression, enzyme function, substrate utilization, mRNA expression(including differential display of whole cell or polyA RNA) and others.

D. In vivo Assays

The present invention particularly contemplates the use of variousanimal models. Transgenic animals may be created with constructs thatpermit MURF expression and activity to be controlled and monitored. Thegeneration of these animals has been described elsewhere in thisdocument.

Treatment of these animals with test compounds will involve theadministration of the compound, in an appropriate form, to the animal.Administration will be by any route the could be utilized for clinicalor non-clinical purposes, including but not limited to oral, nasal,buccal, or even topical. Alternatively, administration may be byintratracheal instillation, bronchial instillation, intradermal,subcutaneous, intramuscular, intraperitoneal or intravenous injection.Specifically contemplated are systemic intravenous injection, regionaladministration via blood or lymph supply.

E. Production of Inhibitors

In an extension of any of the previously described screening assays, thepresent invention also provide for method of producing inhibitors. Themethods comprising any of the preceding screening steps followed by anadditional step of “producing the candidate substance identified as amodulator of the screened activity.

VIII. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1: Material and Methods

Yeast two hybrid screen. Yeast two-hybrid screens of an adult mouseheart cDNA library were performed using full-length serum responsefactor (SRF) fused to the GAL4 DNA binding domain, as describedpreviously (Spencer et al., 1999). Plasmids were isolated andtransformed into electrocompetent XLI-Blue E coli. Plasmids wereprepared by alkaline lysis extraction and sequenced. Sequence analysisand comparison was performed using the NCBI web site Blast program.

Mammalian expression plasmid construction. Full-length MURF-1 expressionplasmids and C-terminal deletion mutants were constructed by PCR usingExpand High Fidelity polymerase (Roche) and cloned in-frame into vectorpcDNA3.1 myc/HIS (Invitrogen) or pECE-Flag. N-terminal deletion mutantswere constructed by PCR, generating an initiator methionine at desiredpositions and cloned into the SacII and HindIII sites of full-lengthMURF-1 in vector pcDNA3.1 myc/HIS. The SacII site is immediatelydownstream of the Kozak translation initiation sequence contained in theMURF-1 cDNA. The Flag-tagged versions of MURF were subcloned intopcDNA3.1 for use in in vitro analyses.

Radioactive in situ hybridization. RNA probes corresponding to the senseand antisense strands of the MURF-1 cDNA were prepared using T7 RNApolymerase (Roche). In situ hybridization was performed as previouslydescribed (Benjamin et al., 1997).

Cell culture, transfection alkaloid treatment, and immunofluorescence.Cos-1, Hela, 10T1/2, 293, 3T3 and 293T cells were maintained in DMEMsupplemented with 10% heat-inactivated fetal bovine serum (FBS). C2cells were maintained in DMEM containing 20% heat-inactivated FBS. Cellswere transfected with 1 μg of various MURF-1 expression plasmids usingFugene6 reagent as recommended by the manufacturer (Roche). 293T cellswere transfected using the calcium phosphate coprecipitation technique(Spencer and Misra, 1996). Cells were treated for 2 hours with 2 μMnocodazole (Sigma) or 2 μM cytochalasin D (Sigma) as indicated.

For immunofluorescence, cells were extracted with 1% Triton X-100 in PBSfor 1 minute, washed twice in PBS, and fixed for 15 minutes in 3.7%formalin in PBS. Cells were then washed three times in PBS and blockedin PBS containing 3% Bovine Serum Albumin and 0.1% NP40 for 30 minutes.All antibodies were incubated in fresh block solution for 60 minutes atambient temperature. Myc-tagged versions of MURF-1 were detected usingantibody 9E10 (Santa Cruz) at a dilution of 1:250. Flag-tagged versionsof MURF-1 were detected using the M2 Flag antibody at a dilution of1:500 (Kodak). Microtubules were detected using alpha-tubulin antibodyat a dilution of 1:2000 (Sigma). Intermediate filaments were visualizedusing vimentin antibody at a dilution of 1:500 (Sigma). Actin wasvisualized using Rhodamine-conjugated phalloidin (Sigma). Secondaryantibodies were purchased from Vector labs and used at a dilution of1:400 in fresh block solution. C2 cells were treated as described aboveexcept the polyclonal antibody UT82, raised against MURF-1, was used ata dilution of 1:500.

In vitro transcription/translation and immunoprecipitation. In vitrotranscription/translation was performed as directed by the manufacturer(Promega). Plasmids used were C-terminally myc-tagged full-length MURF-1and various Flag-tagged deletions of MURF-1. Immunoprecipitations wereperformed using ³⁵S-methionine-labelled in vitro translation products.Briefly, ³⁵S-methionine-labelled products were incubated with agitationin 1 ml of immunoprecipitation buffer (PBS, 0.5% Triton X-100, 0.25 mMzinc sulphate) at 4° C. for 2 hours. 2.5 μl of the Flag-antibody M2 wasadded and incubated an additional 60 minutes. 40 μl of protein A/G-plusbeads (Santa Cruz) were added and incubated 30 minutes. Reactions werecentrifuged at 2500 rpm for 5 minutes at 4° C. Pellets were washed withrocking for 5 minutes in 1 ml of immunoprecipitation buffer. Washing wasrepeated 4 times before resuspension of the pellet in 20 μl ofSDS-sample buffer Approximately 10 μl was used in SDS-PAGE analysis.Gels were dried under vacuum and results were visualized byautoradiography.

Northern blot analysis, RNA isolation and RT-PCR. Northern blot analysiswas performed using a multiple tissue Northern blot from Clontech and³²P-labeled probe made from the MURF-1 full-length cDNA. Hybridizationwas performed for 1 h using rapid-hybe buffer (Amersham). C2 cells wereinduced to differentiate by the addition of DMEM supplemented with 2%horse serum. Cells were incubated for various lengths of time and totalRNA isolated by TriZol extraction (GIBCO). RT-PCR was perfomed asdescribed in (Rawls et al., 1998).

UT 82 Antibody production. An NcoI-HindIII fragment spanning the codingregion for amino acids 271-366 of MURF-1 was fused in-frame into vectorpGEX2T-polyG. BL21-DE3 cells containing the GST-fusion expressionplasmid were grown to an optical density of 0.5 and induced with 1 mMIPTG for 3 h at 30° C. Cells were pelleted and washed in ice cold PBSLysis was induced by sonication and protein was purified by GST-affinitychromatography using standard techniques. Rabbit immunization wasconducted at Cocalico Biological.

Western blot. Whole cell extracts from mouse tissues and C2 cells wereprepared as described previously (Spencer et al. 1999). Approximately 25μg of total protein was used in the analysis. MURF UT82 was used at adilution of 1:4000, anti-Glu-tubulin was used at a dilution of 1:2000,and anti-tubulin was used at a dilution of 1:10,000. Mouse and rabbitHRP-conjugated secondary antibodies were used at a dilution of 1.5000Chemiluminescence was detected using Luminol Reagent (Santa Cruz).

Microtubule sedimentation assays. For endogenous MURF-microtubulecosedimentation experiments, two-month old female mice were euthanizedby standard protocols, hearts and quadriceps (100 mg each tissue) wereremoved, and placed in ice cold PBS containing protease inhibitors(Roche) for 20 minutes. Tissue was finely minced and placed in 1 ml ofPCM buffer (0.1 M Pipes, pH 6.9, 2.5 mM CaCI₂, 1 mM MgSO₄, pH 6.9) plusprotease inhibitors. This was promptly homogenized in a ground glassDounce homogenizer with 25-30 strokes on ice. Nuclei and debris wereremoved by low speed centrifugation (3000 rpm) for 5 minutes at 4° C.The supernatant was centrifuged at 100,000×g for 30 minutes at 4° C. toremove cytoplasmic contaminants. The resulting supernatant was used inthe microtubule sedimentation analysis. The supernatant was supplementedwith 2 mM GTP and 5 mM EGTA and incubated at 37° C. for 20 minutes. Thiswas layered onto a 25% sucrose cushion in PEM buffer (0.1 M Pipes, 1 mMEGTA, 1 mM MgSO₄) plus 1 mM GTP and centrifuged at 20,000×g for 30minutes at room temperature. The supernatant was removed and acetoneprecipitated. The pellet was resuspended in PEM buffer minus GTP andsupplemented to 2 mM CaCl₂. This was placed on ice for 30 min. Thesecond polymerization was induced by adding EGTA and GTP to 5 mM and 1mM, respectively, and incubation at 37° C.Polymerization-depolymerization was repeated 3 times to avoidcytoplasmic contamination. The final polymerization was induced asdescribed above and supplemented with 20 μM taxol (Sigma). The resultingmicrotubule pellet was resuspended in 50 μl of PEM. Approximately 25 μgof protein was used for western analysis. For microtubule sedimentationassays using transfected cells, cells were harvested and treated asdescribed in (Kaufman et al. 1999).

Example 2: Results

Isolation of MURF-1 cDNA. In a two-hybrid screen for cardiac factorsthat interact with serum response factor (SRF), the inventors identifieda cDNA encoding a novel muscle-specific RING-finger protein, which theinventors named MURF-1 (Muscle RING Finger). Subsequent attempts todemonstrate interaction between SRF and MURF-1, or colocalization of theproteins in mammalian cells, were unsuccessful. Thus, the inventors donot believe this interaction is biologically significant, but it mayreflect an ability of SRF to interact with other RING-finger proteins,as several such proteins have been shown to regulate transcription(Hsieh et al. 1999). Nevertheless, because of its interesting structureand expression pattern (see FIG. 2), the inventors continued tocharacterize MURF-1.

The MURF-1 cDNA encodes a 366 amino acid protein with a predictedmolecular weight of 41 kDa and pl of 4.82 (FIG. 1A, Accession number).MURF-1 contains several domains that identify it as a RBCC-type ofRING-finger protein. A RING-finger of the C₃HC₄ type is located near theamino-terminus (amino acids 26-81), followed by another type ofzinc-finger termed a B-box (amino acids 126-158) (FIG. 1B). In all otherRBBC proteins, the spacing between the RING-finger and B-box is alsoabout 40 amino acids (Borden, 1998; Saurin et al., 1996). A predictedleucine-rich coiled-coil domain (amino acids 212-253) and an acidicregion (amino acids 335-366) are located in the C-terminal portion ofthe protein.

Database searches with the amino acid sequence of MURF-1 revealedhighest homology to the Opitz-G/BBB syndrome protein Mid1 and therelated factor Mid2 (FIG. 1C and FIG. 1D) with greatest homology in theRING-finger and B-box domains. Interestingly, MURF-1 does not containthe first B-box of Mid1 and Mid2 nor the butyrophilin-like domain at theC-termini of Mid2 and Mid2, suggesting functional differences betweenthe proteins.

MURF-1 expression is restricted to striated muscle. The expressionpattern of MURF-1 was examined during mouse embryogenesis by in situhybridization. At E8.5, MURF-1 expression was observed only in thedeveloping cardiac region and at E10.5 expression was restrictedexclusively to the heart and the myotome of the somites which gives riseto skeletal muscle. This muscle-specific expression continued throughoutprenatal development, with expression observed in the heart and skeletalmuscle of the intercostals, diaphragm, limbs, face, and head (see FIG.2A).

In adult mice, Northern analysis showed a single MURF-1 transcript ofabout 1.5 kB in cardiac and skeletal muscle (FIG. 2B). Extendedexposures (greater than 6 days) revealed a very low level of expressionin the lung and brain. Consistent with the restricted expression ofMURF-1 mRNA, Western blot analysis of protein from mouse heart,quadriceps, spleen and lung, using anti-MURF antibody, detected MURF-1protein only in heart and skeletal muscle (FIG. 2C). The size of theprotein, 41 kDa, was in agreement with the size predicted from the openreading frame.

The inventors also examined expression of MURF-1 during differentiationof the C2 skeletal muscle cell line. As shown in FIG. 2D, MURF-1transcripts, measured by semi-quantitative RT-PCR, were upregulatedduring myoblast differentiation, in parallel with MyoD and myogenin.Interestingly, MURF-1 expression was observed prior to the expression ofthe muscle structural gene skeletal α-actin, suggesting an early rolefor MURF-1 in myogenesis. This is consistent with expression observed inthe myotome (see FIG. 2A).

Endogenous MURF-1 cosediments with microtubules from striated muscle.Given the amino acid homology between Mid1 and MURF-1 and the ability ofMid1 to interact with microtubules (Cainarca et al., 1999; Schweiger etal. 1999), the inventors investigated whether MURF-1 was also amicrotubule-binding protein. To determine if MURF-1 associates withmicrotubules, microtubule sedimentation assays were performed usingsoluble extracts prepared from mouse skeletal muscle and heart. As seenin FIG. 3, MURF-1 was contained in the microtubule pellet from thesemuscle extracts and was absent from the supernatant, demonstrating aphysical association between C MURF-1 and microtubules in vivo. Nocontaminating actin filaments or intermediate filaments were detected inthe microtubules pellets under these experimental conditions.Interestingly, a small amount of MURF-1 was also contained in theinitial high speed pellet before microtubule polymerization, suggestingits association with structures in addition to microtubules or withcold-stable microtubules that sediment in the initial high speedcentrifugation.

Association of MURF-1 with microtubules is mediated by the leucine-richcoiled-coil domain. To identify the domain of MURF-1 required formicrotubule association, a series of MURF-1 deletion mutants withmyc-tags at their C-termini was constructed and analyzed byimmunofluorescence in Hela cells. As seen in FIG. 4A, MURF-1 wasincorporated into an undulating reticular network in the cytoplasm,reminiscent of the subcellular distribution of Mid1 and Mid2 (Buchner etal., 1999, Cainarca et al., 1999; Schweiger et al., 1999). This networkwas extremely stable, remaining intact even after extraction with 1%Triton X-100. The resistance to extraction in high detergentconcentrations suggested that MURF-1 associated with a cytoskeletalcomponent.

To determine if MURF-1 colocalized with the cytoskeleton,coimmunofluorescence was performed using antibodies specific to actin,vimentin (intermediate filaments) and tubulin. Microtubules showed thestrongest colocalization with MURF-1 (FIG. 4B and C), in agreement withthe results of in vivo microtubule cosedimentation (see FIG. 3). Therewas no overlap of MURF-1 localization with actin and only partialoverlap with intermediate filaments at regions where microtubules andintermediate filaments were in close proximity. Similar localization ofMURF-1 to microtubules was observed in skeletal muscle cells (FIG. 3B).

As shown in FIG. 4D and E, deletion of the 16 amino terminal residues ofMURF-1 (mutant N16) did not disrupt colocalization with microtubules.However, the smooth tubular structures formed with full-length MURF-1were distorted to a thicker and angular structure (FIG. 4D and E). Thesignificance of this redistribution is unclear. Deletion of amino acids1-81 (mutant N81), removing the RING-finger domain, also did not abolishmicrotubule association, but prevented filament formation alongmicrotubules (FIG. 4D and F and see below). Truncation of MURF-1 toamino acid 167 (mutant N167), deleting the B-box domain, resulted insimilar microtubule association without filament formation (FIG. 4D),indicating that the B-box is not involved in microtubule-binding.Deletion of amino acids 1-212 (mutant N212), leaving only theleucine-rich coiled-coil domain and acidic C-terminus, again preventedfilament formation without affecting microtubule binding (FIG. 4D).Instead, as observed with mutants N81 and N167, particulate structureswere formed that colocalized with microtubules. Deletion of theC-terminal acidic region (mutant C31) did not affect the association ofMURF-1 with microtubules nor filament formation (FIG. 4D and G)Truncation of the acidic and coiled-coil region of MURF (mutant C199),leaving only the RING-finger and B-box domains, produced aggregates thatdid not colocalize with microtubules (FIG. 4D). Deletion analyses wereperformed in Cos, 10T1/2, 293 and 3T3 cells with identical results.Taken together, these data demonstrate that the leucine-rich coiled-coilregion of MURF-1 is sufficient for colocalization with microtubules,whereas the RING-finger is required for filament formation alongmicrotubules.

The leucine-rich coiled-coil domain interacts with microtubules. Giventhat MURF-1 cosediments with microtubules and the leucine-richcoiled-coil domain is required for colocalization of MURF-1 withmicrotubules, the inventors determined if this domain also mediatescosedimentation with microtubules. 293T fibroblasts were transfectedwith plasmids expressing wild-type and deletion mutants of MURF-1,simultaneously treated with nocodazole for 24 hours to preventmicrotubule association, and soluble extracts prepared. It was necessaryto treat cells with nocodazole during transfection due to MURF-1′sability to stabilize microtubules. In experiments not employingsimultaneous treatment/transfection, full-length MURF-1, and deletionmutants lacking the N-terminal 16 amino acids (N16) and MURF-1 lackingthe C-terminal 31 amino acids (C31), all shown to form filaments alongmicrotubules, were consistently contained exclusively in the initialhigh speed pellet before microtubule assembly. As seen in FIG. 5C,full-length MURF-1 cosedimented with microtubules in transfected cells.Consistent with the deletion/immunofluorescence analysis (FIG. 4), allMURF-1 deletion mutants containing the leucine-rich coiled-coil domaincosedimented with microtubules. Interestingly, however, mutant N212,which contains only the leucine-rich coiled-coil domain, was alsocontained in the supernatant after microtubule pelleting (FIG. 5C, lane5). Since mutant N167 was localized exclusively to the microtubulepellet, this suggests that although the leucine-rich coiled-coil domainis sufficient for association with microtubules, this domain alsorequires amino acids 167-212 for optimal interaction. Consistent withthe aggregation of mutant C199, containing the RING-finger plus B-box,observed by immunofluorescence, this mutant was consistently containedin the initial high speed pellet before microtubule assembly. These datafurther demonstrate that MURF-1 is capable of cosedimenting withmicrotubules and that the leucine-rich coiled-coil domain is requiredfor this association.

MURF-1 association with microtubules requires homo-oligomerizationmediated by the leucine-rich coiled-coil domain. Coiled-coil domainsoften mediate protein-protein interactions. To determine if theleucine-rich coiled-coil domain might mediate homo-oligomerization ofMURF-1, the inventors performed coimmunoprecipitation experiments using³⁵S-labeled in vitro translated myc-tagged MURF-1 and Flag-taggedMURF-1. As summarized in FIG. 6A, MURF-1 was able to self-associate.Deletion of the RING finger, and B-box (mutants N81 and N167) did notaffect homo-oligomerization, whereas deletion of the leucine-richcoiled-coil domain abolished homo-oligomerization (mutant C199). Thesedata demonstrate that the leucine-rich coiled-coil domain of MURF-1 is ahomo-oligomerization domain. Similarly, the coiled-coil domain of RBCCmembers XNF7, Rfp, and Cbl have also been demonstrated to mediatehomo-oligomerization (Bartkiewicz et al, 1999; Cao et al., 1997; Li etal., 1994).

While constructing epitope-tagged versions of MURF-1, the inventorsobserved that the Flag-tag placed at the N-terminus of MURF-1 completelyinhibited interaction with microtubules, resulting in formation ofprotein aggregates (see FIG. 6B). Therefore, to determine if MURF-1associated with microtubules as a homo-oligomer, the N-terminalFlag-tagged MURF and the C-terminal myc-tagged MURF-1 were transfectedseparately or in combination into Hela cells and immunofluorescence wasperformed. As seen in FIG. 6B, the Flag-tagged MURF-1 did notsignificantly colocalize with microtubules, instead forming largeaggregates. In contrast, co-expression of Flag-MURF-1 and the C-terminalmyc-tagged version resulted in colocalization of both tagged versionalong the same filamentous structures (FIG. 6D). This demonstrates thatMURF-1 associates with microtubules as a homo-oligomer. These data areconsistent with the inability of versions of MURF-1 lacking theleucine-rich coiled-coil homo-oligomerization domain to associate withmicrotubules.

MURF-1 stabilizes microtubules. Recently, Midi was shown to associatewith and stabilize microtubules (Cainarca et al., 1999; Spencer et al.1999). Given the homology of MURF-1 with Mid1 and the ability ofendogenous MURF-1 to colocalize and sediment with microtubules, theinventors determined if MURF-1 was able to stabilize microtubules incultured cells. Cos cells were transfected with MURF-1 expressionplasmid, cultured for 2 hours in the presence of 2 ZM nocodazole,detergent extracted, and MURF-1 expression was examined byimmunofluorescence. As shown in FIGS. 7A-C, MURF-1 preventeddepolymerization of microtubules in the presence of nocodazole. Inuntransfected cells lacking MURF-1, microtubules were completelydepolymerized as indicated by the absence of microtubule staining.Similar results were obtained using cold incubation or calcium treatmentof detergent-extracted cells to destabilize microtubules.

To identify the domain of MURF-1 required for microtubule stabilization,the previously described deletion mutants of MURF-1 were examined fortheir ability to protect microtubules from depolymerization intransfected Cos cells. MURF-1 mutants lacking the N-terminal 16 aminoacids or the C-terminal 31 amino acids (mutants N16 and C31) were ableto protect microtubules from destabilization to a degree comparable tothat of full-length MURF (FIG. 7D). Interestingly, deletion mutants ofMURF-1 lacking the RING-finger did not stabilize microtubules. Thisdemonstrates that the microtubule stabilizing effects of MURF-1 aredependent on the ability to form filaments along microtubules.

MURF-1 causes the formation of glutamic acid-modified microtubules.Microtubules exist in several modified forms. The majority of thetubulin contained within microtubules is tyrosinated at its C-terminus.Tubulin that has been detyrosinated can possess a glutamic acid residueat its C-terminus (Glu-tubulin) or this residue can be removed(delta₂-tubulin). Tubulin can also be acetylated. Microtubules composedof modified tubulin, such as acetylated tubulin, Glu-tubulin anddelta₂-tubulin, have been demonstrated to be more stable than unmodifiedor tyrosinated tubulin. The more stable the microtubule, the longer itexists without depolymerization, and the greater the accumulation ofmodified tubulin within the microtubule. Therefore, the degree ofmodification is indicative of the age of the microtubule. As anothermeasure of MURF-1′s ability to stabilize microtubules, cells weretransfected with MURF-1 and immunostained with antibodies specific tothe glutamic acid-modified form of tubulin (Glu-tubulin). As seen inFIG. 8C, only cells possessing the thick tubular network of MURF-1-boundmicrotubules contain Glu-tubulin, suggesting these microtubules areindeed stable and have existed for a significant period withoutdepolymerization. Identical results were obtained using a delta₂-tubulinantibody.

To determine if MURF-1 expression parallels the formation ofGlu-modified microtubules during skeletal muscle differentiation, C2cells were isolated at various stages of differentiation and Glu-tubulinwas analyzed by western blot analysis. As seen in FIG. 8D, theappearance of Glu-tubulin mirrors the expression of MURF-1 during muscledifferentiation. These data demonstrate that MURF-1/microtubuleinteraction leads to a dramatic shift in microtubule equilibrium from adynamic to a static state and suggests a causative role for MURF-1 inthe establishment of stable microtubules required for the formation ofstriated muscle.

Tissue expresssion of MURF2 and MURF3 in embryonic and adults. As withMURF 1, the inventors determined the localization of expression of MURF2and MURF3 in in situ fluorescence studies. Like MURF1, MURF2 appears tobe expressed primarily in heart tissue, but also in skeletal muscle.Heart expression appears at about E10.5 and increases at E12.5 and E16.5MURF3, in contrast, appears to be expressed selectively in cardiactissue, although like MURF1 and MURF2, its expression increasesthroughout embryogensis.

MURFs are intermediate filament binders. Using antibodies to MURFs andto intermediate filaments such as desmin, vimentin and cytokeratin, theinventors determined that each of MURF1, MURF2 and MURF3 associated withintermediate filaments, indicating a role for MURFs in the stabilizationof these molecules as well. In addition, MURF1 was shown to localize toZ-lines of cultured cardiomyocytes, and MURF3 localized to Z-lines andthe nucleus.

All of the COMPOSITIONS and METHODS disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to theCOMPOSITIONS and METHODS and in the steps or in the sequence of steps ofthe method described herein without departing from the concept, spiritand scope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

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                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 6 <210> SEQ ID NO 1 <211> LENGTH: 1431<212> TYPE: DNA <213> ORGANISM: Mus musculus <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (199)..(1296) <400> SEQUENCE: 1aaggagtgta gacagagtgt ctggaaatag acaggggtga gaggagctgt ta#ggggaagg     60gacaggactc ttccaagagg gagcaatagc cgggatccca agaatccagt ca#gcctaaac    120tgaccgagga agggtgcaca ggcaggggag aaggccaacg acagggccac ag#cgaggcag    180 gctccagagc gccgcggg atg aac ttc acg gtg ggt ttc #aag ccg ctg cta      231                   #  Met Asn Phe Thr Val Gly Phe Lys Pro  #Leu Leu                   #    1               # 5                  # 10ggg gat gcg cac aac atg gac aac ttg gag aa#g cag ctc att tgc ccc      279Gly Asp Ala His Asn Met Asp Asn Leu Glu Ly #s Gln Leu Ile Cys Pro             15      #             20      #             25atc tgc ctg gag atg ttc tcc aag ccc gtg gt#g atc ttg ccc tgc caa      327Ile Cys Leu Glu Met Phe Ser Lys Pro Val Va #l Ile Leu Pro Cys Gln         30          #         35          #         40cac aac ctg tgc cgc aag tgt gcc aac gac gt#c ttc cag gcc tct aat      375His Asn Leu Cys Arg Lys Cys Ala Asn Asp Va #l Phe Gln Ala Ser Asn     45              #     50              #     55cct ctg tgg caa tcc cgg ggc tcc aca acg gt#g tct tca gga gga cgt      423Pro Leu Trp Gln Ser Arg Gly Ser Thr Thr Va #l Ser Ser Gly Gly Arg 60                  # 65                  # 70                  # 75ttc cga tgc cca tct tgt agg cac gag gtt gt#c ctg gac agg cat ggt      471Phe Arg Cys Pro Ser Cys Arg His Glu Val Va #l Leu Asp Arg His Gly                 80  #                 85  #                 90gtc tat ggc ctg cag cgg aac ctg cta gtg ga#g aac atc att gac atc      519Val Tyr Gly Leu Gln Arg Asn Leu Leu Val Gl #u Asn Ile Ile Asp Ile             95      #            100      #            105tac aag cag gag tcc tcc cgg cca ctg cac gc#c aag gct gaa cag cac      567Tyr Lys Gln Glu Ser Ser Arg Pro Leu His Al #a Lys Ala Glu Gln His        110           #       115           #       120ctc atg tgt gag gag cac gag gac gag aag at#c aac atc tac tgc ctg      615Leu Met Cys Glu Glu His Glu Asp Glu Lys Il #e Asn Ile Tyr Cys Leu    125               #   130               #   135agc tgc gag gtg ccc acc tgc tct ctc tgc aa#g gtt ttc ggc gcc cac      663Ser Cys Glu Val Pro Thr Cys Ser Leu Cys Ly #s Val Phe Gly Ala His140                 1 #45                 1 #50                 1 #55aag gac tgt gag gtg gcc cct ctg ccc acc at#t tac aaa cgc cag aag      711Lys Asp Cys Glu Val Ala Pro Leu Pro Thr Il #e Tyr Lys Arg Gln Lys                160   #               165   #               170agt gag ctg agc gat ggc atc gcg atg ctg gt#g gcg ggc aat gac cgt      759Ser Glu Leu Ser Asp Gly Ile Ala Met Leu Va #l Ala Gly Asn Asp Arg            175       #           180       #           185gtg cag gca gtg atc acc cag atg gag gag gt#g tgc cag acc att gag      807Val Gln Ala Val Ile Thr Gln Met Glu Glu Va #l Cys Gln Thr Ile Glu        190           #       195           #       200gac aac agc cgc aga cag aag caa ctg tta aa#c cag agg ttc gag acc      855Asp Asn Ser Arg Arg Gln Lys Gln Leu Leu As #n Gln Arg Phe Glu Thr    205               #   210               #   215ctg tgc gcg gtt ttg gag gag cgc aag ggc ga#a ctg ctt caa gca ctg      903Leu Cys Ala Val Leu Glu Glu Arg Lys Gly Gl #u Leu Leu Gln Ala Leu220                 2 #25                 2 #30                 2 #35gcc cgg gag cag gag gag aag ttg cag cgc gt#g cgg ggc ctc atc cgc      951Ala Arg Glu Gln Glu Glu Lys Leu Gln Arg Va #l Arg Gly Leu Ile Arg                240   #               245   #               250cag tac gga gac cac ttg gag ggc tcc tca aa#g ctg gtg gag tcc gcc      999Gln Tyr Gly Asp His Leu Glu Gly Ser Ser Ly #s Leu Val Glu Ser Ala            255       #           260       #           265atc cag tcc atg gag gag ccg cag atg gct ct#c tac ctc cag cag gca     1047Ile Gln Ser Met Glu Glu Pro Gln Met Ala Le #u Tyr Leu Gln Gln Ala        270           #       275           #       280aag gag ctg atc aac aag gtc ggg gca atg tc#g aag gtg gag ctg gca     1095Lys Glu Leu Ile Asn Lys Val Gly Ala Met Se #r Lys Val Glu Leu Ala    285               #   290               #   295gga cgg ccg gag cca ggc tat gag agc atg ga#g caa ttc tct gtg agc     1143Gly Arg Pro Glu Pro Gly Tyr Glu Ser Met Gl #u Gln Phe Ser Val Ser300                 3 #05                 3 #10                 3 #15gtg gag cac gtg gcc gaa atg ttg cga acc at#c gac ttc cag ccg ggc     1191Val Glu His Val Ala Glu Met Leu Arg Thr Il #e Asp Phe Gln Pro Gly                320   #               325   #               330gcc gct ggg gat gaa gag gat gac gac atg gc#t ttg gat ggg gag gag     1239Ala Ala Gly Asp Glu Glu Asp Asp Asp Met Al #a Leu Asp Gly Glu Glu            335       #           340       #           345ggc aat gcg ggg ctg gag gag gag cgg ctg ga#c gtg cca gaa ggc tca     1287Gly Asn Ala Gly Leu Glu Glu Glu Arg Leu As #p Val Pro Glu Gly Ser        350           #       355           #       360ggc ctg cac tgacccgact ctgatccaga gcgcacaccc gaagcggga#g             1336 Gly Leu His     365ccaagggatg ctgaggatct gcgcagagac caccgcgcca ccaagctcgg ct#tcccgccc   1396 ccgggaaggt tctcaataaa ggactcaagt gtccc       #                   #     1431 <210> SEQ ID NO 2 <211> LENGTH: 366<212> TYPE: PRT <213> ORGANISM: Mus musculus <400> SEQUENCE: 2Met Asn Phe Thr Val Gly Phe Lys Pro Leu Le #u Gly Asp Ala His Asn  1               5  #                 10  #                 15Met Asp Asn Leu Glu Lys Gln Leu Ile Cys Pr #o Ile Cys Leu Glu Met             20      #             25      #             30Phe Ser Lys Pro Val Val Ile Leu Pro Cys Gl #n His Asn Leu Cys Arg         35          #         40          #         45Lys Cys Ala Asn Asp Val Phe Gln Ala Ser As #n Pro Leu Trp Gln Ser     50              #     55              #     60Arg Gly Ser Thr Thr Val Ser Ser Gly Gly Ar #g Phe Arg Cys Pro Ser 65                  # 70                  # 75                  # 80Cys Arg His Glu Val Val Leu Asp Arg His Gl #y Val Tyr Gly Leu Gln                 85  #                 90  #                 95Arg Asn Leu Leu Val Glu Asn Ile Ile Asp Il #e Tyr Lys Gln Glu Ser            100       #           105       #           110Ser Arg Pro Leu His Ala Lys Ala Glu Gln Hi #s Leu Met Cys Glu Glu        115           #       120           #       125His Glu Asp Glu Lys Ile Asn Ile Tyr Cys Le #u Ser Cys Glu Val Pro    130               #   135               #   140Thr Cys Ser Leu Cys Lys Val Phe Gly Ala Hi #s Lys Asp Cys Glu Val145                 1 #50                 1 #55                 1 #60Ala Pro Leu Pro Thr Ile Tyr Lys Arg Gln Ly #s Ser Glu Leu Ser Asp                165   #               170   #               175Gly Ile Ala Met Leu Val Ala Gly Asn Asp Ar #g Val Gln Ala Val Ile            180       #           185       #           190Thr Gln Met Glu Glu Val Cys Gln Thr Ile Gl #u Asp Asn Ser Arg Arg        195           #       200           #       205Gln Lys Gln Leu Leu Asn Gln Arg Phe Glu Th #r Leu Cys Ala Val Leu    210               #   215               #   220Glu Glu Arg Lys Gly Glu Leu Leu Gln Ala Le #u Ala Arg Glu Gln Glu225                 2 #30                 2 #35                 2 #40Glu Lys Leu Gln Arg Val Arg Gly Leu Ile Ar #g Gln Tyr Gly Asp His                245   #               250   #               255Leu Glu Gly Ser Ser Lys Leu Val Glu Ser Al #a Ile Gln Ser Met Glu            260       #           265       #           270Glu Pro Gln Met Ala Leu Tyr Leu Gln Gln Al #a Lys Glu Leu Ile Asn        275           #       280           #       285Lys Val Gly Ala Met Ser Lys Val Glu Leu Al #a Gly Arg Pro Glu Pro    290               #   295               #   300Gly Tyr Glu Ser Met Glu Gln Phe Ser Val Se #r Val Glu His Val Ala305                 3 #10                 3 #15                 3 #20Glu Met Leu Arg Thr Ile Asp Phe Gln Pro Gl #y Ala Ala Gly Asp Glu                325   #               330   #               335Glu Asp Asp Asp Met Ala Leu Asp Gly Glu Gl #u Gly Asn Ala Gly Leu            340       #           345       #           350Glu Glu Glu Arg Leu Asp Val Pro Glu Gly Se #r Gly Leu His        355           #       360           #       365<210> SEQ ID NO 3 <211> LENGTH: 2590 <212> TYPE: DNA<213> ORGANISM: Mus musculus <220> FEATURE: <221> NAME/KEY: CDS<222> LOCATION: (80)..(1714) <400> SEQUENCE: 3ctcgagattt acccttacag aagctgttcg ggagcacctt tcccttggca gc#acactcag     60 ggacagggac ggcaaggaa atg agc act tct ctg aat tac# aag tct ttc tcc     112                   #   Met Ser Thr Ser Leu Asn Tyr Lys Ser # Phe Ser                   #     1              #  5                 #  10aaa gag cag cag acc atg gat aac ttg gaa aa#g caa ctg atc tgt ccc      160Lys Glu Gln Gln Thr Met Asp Asn Leu Glu Ly #s Gln Leu Ile Cys Pro             15      #             20      #             25atc tgc cta gag atg ttc acg aag cct gtg gt#c att ctc cct tgc cag      208Ile Cys Leu Glu Met Phe Thr Lys Pro Val Va #l Ile Leu Pro Cys Gln         30          #         35          #         40cac aac ctg tgc agg aaa tgt gcc agt gac at#c ttc cag gcc tct aac      256His Asn Leu Cys Arg Lys Cys Ala Ser Asp Il #e Phe Gln Ala Ser Asn     45              #     50              #     55ccg tac tta ccc aca aga gga ggc acc acc gt#g gca tca ggg ggc cgc      304Pro Tyr Leu Pro Thr Arg Gly Gly Thr Thr Va #l Ala Ser Gly Gly Arg 60                  # 65                  # 70                  # 75ttc cgc tgt ccc tcc tgc aga cat gag gtg gt#g tta gac aga cat ggg      352Phe Arg Cys Pro Ser Cys Arg His Glu Val Va #l Leu Asp Arg His Gly                 80  #                 85  #                 90gtc tat gga ctg cag agg aac ctg ctc gtg ga#a aac att att gat atc      400Val Tyr Gly Leu Gln Arg Asn Leu Leu Val Gl #u Asn Ile Ile Asp Ile             95      #            100      #            105tac aag cag gaa tcc acc agg cca gaa aaa aa#a ttg gac cag ccc atg      448Tyr Lys Gln Glu Ser Thr Arg Pro Glu Lys Ly #s Leu Asp Gln Pro Met        110           #       115           #       120tgt gaa gag cat gaa gag gaa cgc atc aac at#c tat tgt ctg aac tgt      496Cys Glu Glu His Glu Glu Glu Arg Ile Asn Il #e Tyr Cys Leu Asn Cys    125               #   130               #   135gaa gtg ccc acc tgt tcc ttg tgc aag gtt tt#t ggc gcc cat aag gac      544Glu Val Pro Thr Cys Ser Leu Cys Lys Val Ph #e Gly Ala His Lys Asp140                 1 #45                 1 #50                 1 #55tgc cag gtg gct ccc ctg act cat gtg ttc ca#g agg cag aag tca gag      592Cys Gln Val Ala Pro Leu Thr His Val Phe Gl #n Arg Gln Lys Ser Glu                160   #               165   #               170ctc agt gat ggt att gct gta ctt gtg gga ag#c aac gat aga gtc cag      640Leu Ser Asp Gly Ile Ala Val Leu Val Gly Se #r Asn Asp Arg Val Gln            175       #           180       #           185ggt gtg atc agc cag ctg gag gac acc tgt aa#a act att gag gag tgc      688Gly Val Ile Ser Gln Leu Glu Asp Thr Cys Ly #s Thr Ile Glu Glu Cys        190           #       195           #       200tgc aga aag cag aaa cag gac ctg tgt gag aa#a ttt gat cac cta tac      736Cys Arg Lys Gln Lys Gln Asp Leu Cys Glu Ly #s Phe Asp His Leu Tyr    205               #   210               #   215ggc atc ctg gag gag agg aag act gaa atg ac#c caa gcc atc act cga      784Gly Ile Leu Glu Glu Arg Lys Thr Glu Met Th #r Gln Ala Ile Thr Arg220                 2 #25                 2 #30                 2 #35aca cag gag gag aaa ctg gaa cat gtc cga ac#t ctt atc agg aag tat      832Thr Gln Glu Glu Lys Leu Glu His Val Arg Th #r Leu Ile Arg Lys Tyr                240   #               245   #               250tcc gat cac ctg gag aac gta tcc aag ttg gt#g gag tca gga atc cag      880Ser Asp His Leu Glu Asn Val Ser Lys Leu Va #l Glu Ser Gly Ile Gln            255       #           260       #           265ttc atg gat gag ccc gaa atg gca gta ttt ct#g cag aat gcc aag acc      928Phe Met Asp Glu Pro Glu Met Ala Val Phe Le #u Gln Asn Ala Lys Thr        270           #       275           #       280ctg ttg caa aag atc gtg gaa gca tca aag gc#g ttt cag atg gag aaa      976Leu Leu Gln Lys Ile Val Glu Ala Ser Lys Al #a Phe Gln Met Glu Lys    285               #   290               #   295cta gaa caa ggt tat gag atc atg agc aac tt#c act gtc aat ctc aat     1024Leu Glu Gln Gly Tyr Glu Ile Met Ser Asn Ph #e Thr Val Asn Leu Asn300                 3 #05                 3 #10                 3 #15aga gaa gaa aaa att atc cgt gaa att gac tt#t tct aga gaa gag gaa     1072Arg Glu Glu Lys Ile Ile Arg Glu Ile Asp Ph #e Ser Arg Glu Glu Glu                320   #               325   #               330gag gaa gaa gat gca gga gaa ata gat gaa ga#a gga gaa gga gag gat     1120Glu Glu Glu Asp Ala Gly Glu Ile Asp Glu Gl #u Gly Glu Gly Glu Asp            335       #           340       #           345gca gta gaa gta gaa gag gca gaa aat gtt ca#a ata gca tct tca ggg     1168Ala Val Glu Val Glu Glu Ala Glu Asn Val Gl #n Ile Ala Ser Ser Gly        350           #       355           #       360gaa gag gag agt ctg gag aaa gct gca gag cc#c tct cag ctt ccc gca     1216Glu Glu Glu Ser Leu Glu Lys Ala Ala Glu Pr #o Ser Gln Leu Pro Ala    365               #   370               #   375gag ctt cag gtc gcc cca gag cca cta cct gc#t tcc tct cca gaa ccg     1264Glu Leu Gln Val Ala Pro Glu Pro Leu Pro Al #a Ser Ser Pro Glu Pro380                 3 #85                 3 #90                 3 #95ttt tca tcc atg cca cct gct gca gat gtc ct#g gtg aca cag ggg gag     1312Phe Ser Ser Met Pro Pro Ala Ala Asp Val Le #u Val Thr Gln Gly Glu                400   #               405   #               410gtg gtg ccc att ggc tct cag cag acc aca ca#g tct gaa act tca ggc     1360Val Val Pro Ile Gly Ser Gln Gln Thr Thr Gl #n Ser Glu Thr Ser Gly            415       #           420       #           425cct tca gca gcg gaa act gcg gat ccc ttg tt#t tac cct agt tgg tat     1408Pro Ser Ala Ala Glu Thr Ala Asp Pro Leu Ph #e Tyr Pro Ser Trp Tyr        430           #       435           #       440aaa ggc caa agc cgg aaa acc agc tcc aac cc#a cct tgc act cat ggg     1456Lys Gly Gln Ser Arg Lys Thr Ser Ser Asn Pr #o Pro Cys Thr His Gly    445               #   450               #   455agt gaa ggt ctg ggt caa ata ggg cct ctg gg#c att gag gat tcc agt     1504Ser Glu Gly Leu Gly Gln Ile Gly Pro Leu Gl #y Ile Glu Asp Ser Ser460                 4 #65                 4 #70                 4 #75gtg cag tcc gca gaa gtg gca gaa gcc gca ac#c aat gag cag gca gca     1552Val Gln Ser Ala Glu Val Ala Glu Ala Ala Th #r Asn Glu Gln Ala Ala                480   #               485   #               490gtg agt ggt aag gag tct agt tca act gca gc#t acc tct cag att gga     1600Val Ser Gly Lys Glu Ser Ser Ser Thr Ala Al #a Thr Ser Gln Ile Gly            495       #           500       #           505ttt gag gcc cct tct ccc cag gga cag tct gc#a gcc ttg ggg agt ggg     1648Phe Glu Ala Pro Ser Pro Gln Gly Gln Ser Al #a Ala Leu Gly Ser Gly        510           #       515           #       520ggt ggg gtg atc ctg agc cag ctc gcc acg tc#t tct cct tct cct ggt     1696Gly Gly Val Ile Leu Ser Gln Leu Ala Thr Se #r Ser Pro Ser Pro Gly    525               #   530               #   535ttg aat tcc cta aat gaa taatatttat tctcgttgct gc#cccctgtc            1744 Leu Asn Ser Leu Asn Glu 540                 5#45 tgcctggctg aaaagcacat aggcagcagg aaacaggtgg aaattcacca cg#attcatat   1804gaaggggacc tctggacagg atttctgaaa gcaaaacaaa acaatacaac ac#caccaccc   1864tttaattcca gatgacttat ctcactcatt gagaaaatga ttatgctcag aa#caaaatta   1924cagaaaatac tcttctgaag aaacttgatc ttctgcaaat ctttcatttg tg#tgagaaac   1984cttctgaagg ttgtgtaggt gtggtgcatg cctgtgtatc agccataagt gc#caagtggt   2044aacaaagtgg cagaacactc tcccagcctc cctcaggctt ctggttattt ta#ggacgctt   2104gtgccttttg cttttctcct tagcattgca ggtggtaggt gatgttcagt gt#cagttcca   2164aactgaccga tttatcaaaa tatggagatt ggtcactgac caaagctatg ta#gggcactg   2224tagaggttcc tttccctatg gatgccatgg gtgcgcagac aggactttcc tt#tacatgtg   2284gccacacgtc catagtccag aaggccaaaa atctagggca actcttttga ca#tttttcta   2344accttattta catatctcat aatcatatcc atgtattagg cattttaatt ga#atttcaaa   2404gaggagctgt ctactttctt aagtgtcctg ccatagcagc aatctgataa tc#tgtggagc   2464aactgcatgg atttaagtat acacacaatt ctccccctgt gtgccttctc tc#tctctctc   2524tctccccctc tctccctctg tctcttctct ccccctctgt ctctccctcc tt#tcctttct   2584 tcctcc                  #                  #                   #         2590 <210> SEQ ID NO 4 <211> LENGTH: 545<212> TYPE: PRT <213> ORGANISM: Mus musculus <400> SEQUENCE: 4Met Ser Thr Ser Leu Asn Tyr Lys Ser Phe Se #r Lys Glu Gln Gln Thr  1               5  #                 10  #                 15Met Asp Asn Leu Glu Lys Gln Leu Ile Cys Pr #o Ile Cys Leu Glu Met             20      #             25      #             30Phe Thr Lys Pro Val Val Ile Leu Pro Cys Gl #n His Asn Leu Cys Arg         35          #         40          #         45Lys Cys Ala Ser Asp Ile Phe Gln Ala Ser As #n Pro Tyr Leu Pro Thr     50              #     55              #     60Arg Gly Gly Thr Thr Val Ala Ser Gly Gly Ar #g Phe Arg Cys Pro Ser 65                  # 70                  # 75                  # 80Cys Arg His Glu Val Val Leu Asp Arg His Gl #y Val Tyr Gly Leu Gln                 85  #                 90  #                 95Arg Asn Leu Leu Val Glu Asn Ile Ile Asp Il #e Tyr Lys Gln Glu Ser            100       #           105       #           110Thr Arg Pro Glu Lys Lys Leu Asp Gln Pro Me #t Cys Glu Glu His Glu        115           #       120           #       125Glu Glu Arg Ile Asn Ile Tyr Cys Leu Asn Cy #s Glu Val Pro Thr Cys    130               #   135               #   140Ser Leu Cys Lys Val Phe Gly Ala His Lys As #p Cys Gln Val Ala Pro145                 1 #50                 1 #55                 1 #60Leu Thr His Val Phe Gln Arg Gln Lys Ser Gl #u Leu Ser Asp Gly Ile                165   #               170   #               175Ala Val Leu Val Gly Ser Asn Asp Arg Val Gl #n Gly Val Ile Ser Gln            180       #           185       #           190Leu Glu Asp Thr Cys Lys Thr Ile Glu Glu Cy #s Cys Arg Lys Gln Lys        195           #       200           #       205Gln Asp Leu Cys Glu Lys Phe Asp His Leu Ty #r Gly Ile Leu Glu Glu    210               #   215               #   220Arg Lys Thr Glu Met Thr Gln Ala Ile Thr Ar #g Thr Gln Glu Glu Lys225                 2 #30                 2 #35                 2 #40Leu Glu His Val Arg Thr Leu Ile Arg Lys Ty #r Ser Asp His Leu Glu                245   #               250   #               255Asn Val Ser Lys Leu Val Glu Ser Gly Ile Gl #n Phe Met Asp Glu Pro            260       #           265       #           270Glu Met Ala Val Phe Leu Gln Asn Ala Lys Th #r Leu Leu Gln Lys Ile        275           #       280           #       285Val Glu Ala Ser Lys Ala Phe Gln Met Glu Ly #s Leu Glu Gln Gly Tyr    290               #   295               #   300Glu Ile Met Ser Asn Phe Thr Val Asn Leu As #n Arg Glu Glu Lys Ile305                 3 #10                 3 #15                 3 #20Ile Arg Glu Ile Asp Phe Ser Arg Glu Glu Gl #u Glu Glu Glu Asp Ala                325   #               330   #               335Gly Glu Ile Asp Glu Glu Gly Glu Gly Glu As #p Ala Val Glu Val Glu            340       #           345       #           350Glu Ala Glu Asn Val Gln Ile Ala Ser Ser Gl #y Glu Glu Glu Ser Leu        355           #       360           #       365Glu Lys Ala Ala Glu Pro Ser Gln Leu Pro Al #a Glu Leu Gln Val Ala    370               #   375               #   380Pro Glu Pro Leu Pro Ala Ser Ser Pro Glu Pr #o Phe Ser Ser Met Pro385                 3 #90                 3 #95                 4 #00Pro Ala Ala Asp Val Leu Val Thr Gln Gly Gl #u Val Val Pro Ile Gly                405   #               410   #               415Ser Gln Gln Thr Thr Gln Ser Glu Thr Ser Gl #y Pro Ser Ala Ala Glu            420       #           425       #           430Thr Ala Asp Pro Leu Phe Tyr Pro Ser Trp Ty #r Lys Gly Gln Ser Arg        435           #       440           #       445Lys Thr Ser Ser Asn Pro Pro Cys Thr His Gl #y Ser Glu Gly Leu Gly    450               #   455               #   460Gln Ile Gly Pro Leu Gly Ile Glu Asp Ser Se #r Val Gln Ser Ala Glu465                 4 #70                 4 #75                 4 #80Val Ala Glu Ala Ala Thr Asn Glu Gln Ala Al #a Val Ser Gly Lys Glu                485   #               490   #               495Ser Ser Ser Thr Ala Ala Thr Ser Gln Ile Gl #y Phe Glu Ala Pro Ser            500       #           505       #           510Pro Gln Gly Gln Ser Ala Ala Leu Gly Ser Gl #y Gly Gly Val Ile Leu        515           #       520           #       525Ser Gln Leu Ala Thr Ser Ser Pro Ser Pro Gl #y Leu Asn Ser Leu Asn    530               #   535               #   540 Glu 545<210> SEQ ID NO 5 <211> LENGTH: 1597 <212> TYPE: DNA<213> ORGANISM: Mus musculus <220> FEATURE: <221> NAME/KEY: CDS<222> LOCATION: (299)..(1327) <400> SEQUENCE: 5ctcgagattt acccttacag aagctgttcg ggagcacctt tcccttggca gc#acactcag     60ggacagggac ggcaaggaaa tgagcacttc tctgaattac aagtctttct cc#aaagagca    120gcagaccatg gataacttgg aaaagcaact gatctgtccc atctgcctag ag#atgttcac    180gaagcctgtg gtcattctcc cttgccagca caacctgtgc aggaaatgtg cg#ggcccccc    240ttggagacaa agacttggtg tgacgcaggt gggcaagaca gtcgcatttc aa#agcaat      298 atg gat tat aaa tct agc ctg att cct gat gg#a aac gct atg gag aac      346Met Asp Tyr Lys Ser Ser Leu Ile Pro Asp Gl #y Asn Ala Met Glu Asn  1               5  #                 10  #                 15ctg gag aag cag ctg atc tgc ccc atc tgc ct#g gag atg ttt acc aag      394Leu Glu Lys Gln Leu Ile Cys Pro Ile Cys Le #u Glu Met Phe Thr Lys             20      #             25      #             30cct gtg gtc atc ctg ccc tgc caa cac aac ct#c tgc cgg aag tgt gcc      442Pro Val Val Ile Leu Pro Cys Gln His Asn Le #u Cys Arg Lys Cys Ala         35          #         40          #         45aac gac atc ttc cag gct gcg aat ccc tac tg#g acc aac cgc ggt ggc      490Asn Asp Ile Phe Gln Ala Ala Asn Pro Tyr Tr #p Thr Asn Arg Gly Gly     50              #     55              #     60tca gtg tcc atg tct gga ggt cgt ttc cgt tg#c ccc tcg tgc cgc cat      538Ser Val Ser Met Ser Gly Gly Arg Phe Arg Cy #s Pro Ser Cys Arg His 65                  # 70                  # 75                  # 80gaa gtg atc atg gac cgg cac ggg gtg tac gg#c ctg cag agg aac ctg      586Glu Val Ile Met Asp Arg His Gly Val Tyr Gl #y Leu Gln Arg Asn Leu                 85  #                 90  #                 95ctg gtg gaa aac atc att gac atc tac aag ca#g gag tgc tcc agt cgg      634Leu Val Glu Asn Ile Ile Asp Ile Tyr Lys Gl #n Glu Cys Ser Ser Arg            100       #           105       #           110ccc ctg cag aaa ggc agc cac ccg atg tgc aa#g gaa cac gaa gac gag      682Pro Leu Gln Lys Gly Ser His Pro Met Cys Ly #s Glu His Glu Asp Glu        115           #       120           #       125aag atc aac atc tac tgt ctc acg tgt gag gt#g cct act tgc tcc ttg      730Lys Ile Asn Ile Tyr Cys Leu Thr Cys Glu Va #l Pro Thr Cys Ser Leu    130               #   135               #   140tgc aag gtg ttt ggg gct cac cag gcc tgt ga#g gtt gcc cct ttg caa      778Cys Lys Val Phe Gly Ala His Gln Ala Cys Gl #u Val Ala Pro Leu Gln145                 1 #50                 1 #55                 1 #60agc atc ttc caa gga cag aag act gag ctg ag#t aac tgc atc tcc atg      826Ser Ile Phe Gln Gly Gln Lys Thr Glu Leu Se #r Asn Cys Ile Ser Met                165   #               170   #               175ctg gtg gcg ggg aac gac cga gtg cag acg at#c atc tct cag ctg gag      874Leu Val Ala Gly Asn Asp Arg Val Gln Thr Il #e Ile Ser Gln Leu Glu            180       #           185       #           190gac tcg tgc aga gtg acc aag gag aat agc ca#c cag gtg aag gag gag      922Asp Ser Cys Arg Val Thr Lys Glu Asn Ser Hi #s Gln Val Lys Glu Glu        195           #       200           #       205ctg agt cag aag ttt gac acc ctc tac gcc at#c ctg gat gag aag aag      970Leu Ser Gln Lys Phe Asp Thr Leu Tyr Ala Il #e Leu Asp Glu Lys Lys    210               #   215               #   220agc gag ctg ctg cag cgg atc acg cag gag ca#g gag gag aag ctg ggc     1018Ser Glu Leu Leu Gln Arg Ile Thr Gln Glu Gl #n Glu Glu Lys Leu Gly225                 2 #30                 2 #35                 2 #40ttc atc gag gct ctg atc ctc cag tac agg ga#g cag ctg gaa aag tcc     1066Phe Ile Glu Ala Leu Ile Leu Gln Tyr Arg Gl #u Gln Leu Glu Lys Ser                245   #               250   #               255acc aag ctt gtg gag acc gcc atc cag tcc ct#g gat gag ccc gga ggg     1114Thr Lys Leu Val Glu Thr Ala Ile Gln Ser Le #u Asp Glu Pro Gly Gly            260       #           265       #           270gct acc ttc ctc tca agt gcc aag cag ctc at#c aag agc att gta gaa     1162Ala Thr Phe Leu Ser Ser Ala Lys Gln Leu Il #e Lys Ser Ile Val Glu        275           #       280           #       285gcc tcc aag ggc tgc cag ctg ggg aag aca ga#g caa ggc ttt gag aac     1210Ala Ser Lys Gly Cys Gln Leu Gly Lys Thr Gl #u Gln Gly Phe Glu Asn    290               #   295               #   300atg gac tac ttt act ctg gac tta gaa cac at#a gca gag gcc ttg agg     1258Met Asp Tyr Phe Thr Leu Asp Leu Glu His Il #e Ala Glu Ala Leu Arg305                 3 #10                 3 #15                 3 #20gcc att gac ttt ggg aca ggt aaa gga tgt ga#t gtt aca tgt ttg acc     1306Ala Ile Asp Phe Gly Thr Gly Lys Gly Cys As #p Val Thr Cys Leu Thr                325   #               330   #               335ttt gaa agg cag cgt tcc tct tgagttctga ggggaactg#t taaaaaagtc        1357 Phe Glu Arg Gln Arg Ser Ser             340aaatttacac agccagtgtt gacaggtctc tctatggagc cctgactgtc tt#agtagtgt   1417ctaagtagac caagctggtc tggaacacat agagatctat cttgcccatc tc#tgcttctt   1477gagggatgag ataaaaggca tgtgcccacc atgcctggct ccacagacaa ct#ttgtgatg   1537gatccagggt ctggcacagt gcctggtaca taattgtttc gaaataaatt at#ctcgtgcc   1597 <210> SEQ ID NO 6 <211> LENGTH: 343 <212> TYPE: PRT<213> ORGANISM: Mus musculus <400> SEQUENCE: 6Met Asp Tyr Lys Ser Ser Leu Ile Pro Asp Gl #y Asn Ala Met Glu Asn  1               5  #                 10  #                 15Leu Glu Lys Gln Leu Ile Cys Pro Ile Cys Le #u Glu Met Phe Thr Lys             20      #             25      #             30Pro Val Val Ile Leu Pro Cys Gln His Asn Le #u Cys Arg Lys Cys Ala         35          #         40          #         45Asn Asp Ile Phe Gln Ala Ala Asn Pro Tyr Tr #p Thr Asn Arg Gly Gly     50              #     55              #     60Ser Val Ser Met Ser Gly Gly Arg Phe Arg Cy #s Pro Ser Cys Arg His 65                  # 70                  # 75                  # 80Glu Val Ile Met Asp Arg His Gly Val Tyr Gl #y Leu Gln Arg Asn Leu                 85  #                 90  #                 95Leu Val Glu Asn Ile Ile Asp Ile Tyr Lys Gl #n Glu Cys Ser Ser Arg            100       #           105       #           110Pro Leu Gln Lys Gly Ser His Pro Met Cys Ly #s Glu His Glu Asp Glu        115           #       120           #       125Lys Ile Asn Ile Tyr Cys Leu Thr Cys Glu Va #l Pro Thr Cys Ser Leu    130               #   135               #   140Cys Lys Val Phe Gly Ala His Gln Ala Cys Gl #u Val Ala Pro Leu Gln145                 1 #50                 1 #55                 1 #60Ser Ile Phe Gln Gly Gln Lys Thr Glu Leu Se #r Asn Cys Ile Ser Met                165   #               170   #               175Leu Val Ala Gly Asn Asp Arg Val Gln Thr Il #e Ile Ser Gln Leu Glu            180       #           185       #           190Asp Ser Cys Arg Val Thr Lys Glu Asn Ser Hi #s Gln Val Lys Glu Glu        195           #       200           #       205Leu Ser Gln Lys Phe Asp Thr Leu Tyr Ala Il #e Leu Asp Glu Lys Lys    210               #   215               #   220Ser Glu Leu Leu Gln Arg Ile Thr Gln Glu Gl #n Glu Glu Lys Leu Gly225                 2 #30                 2 #35                 2 #40Phe Ile Glu Ala Leu Ile Leu Gln Tyr Arg Gl #u Gln Leu Glu Lys Ser                245   #               250   #               255Thr Lys Leu Val Glu Thr Ala Ile Gln Ser Le #u Asp Glu Pro Gly Gly            260       #           265       #           270Ala Thr Phe Leu Ser Ser Ala Lys Gln Leu Il #e Lys Ser Ile Val Glu        275           #       280           #       285Ala Ser Lys Gly Cys Gln Leu Gly Lys Thr Gl #u Gln Gly Phe Glu Asn    290               #   295               #   300Met Asp Tyr Phe Thr Leu Asp Leu Glu His Il #e Ala Glu Ala Leu Arg305                 3 #10                 3 #15                 3 #20Ala Ile Asp Phe Gly Thr Gly Lys Gly Cys As #p Val Thr Cys Leu Thr                325   #               330   #               335Phe Glu Arg Gln Arg Ser Ser             340

What is claimed is:
 1. An isolated DNA segment encoding a MURF-1polypeptide either having, (i) the amino acid sequence set forth in SEQID NO:2, or, (ii) a variant of the amino acid sequence set forth in SEQID NO:2 capable of binding a microtubule wherein the variant is encodedby a nucleic acid sequence that hybridizes to SEQ ID NO:1, from position199 through position 1296, inclusive, under conditions of 10 mM Tris-HCl(pH 8.3), 50 mM KCl, and 1.5 μM MgCl₂ at a temperature of 72° C.
 2. TheDNA segment of claim 1, wherein the MURF-1 polypeptide has the sequenceof SEQ ID NO:2.
 3. The DNA segment of claim 2, wherein the MURF-1 DNAsegment has the sequence of SEQ ID NO:1.
 4. The DNA segment of claim 1,wherein the DNA segment is positioned under the control of a promoter.5. The DNA segment of claim 4, wherein the promoter is not a nativeMURF-1, MURF-2 or MURF-3 promoter.
 6. The DNA segment of claim 4,further comprising a polyadenylation signal.
 7. The DNA segment of claim4, further comprising an origin of replication.
 8. The DNA segment ofclaim 7, wherein the DNA segment is comprised within a viral vector. 9.The DNA segment of claim 8, wherein the DNA segment is comprised withina non-viral vector.
 10. A host cell comprising a DNA segment of claim 1wherein said DNA segment comprises a promoter heterologous to the murineMURF-1 polypeptide coding region set forth in SEQ ID NO:1.
 11. The hostcell of claim 10, wherein the MURF-1 polypeptide has the sequence of SEQID NO:2.
 12. The host cell of claim 10, further defined as a prokaryotichost cell.
 13. The host cell of claim 10, further defined as aeukaryotic host cell.
 14. The host cell of claim 13, wherein the hostcell is a secretory cell.
 15. A method of producing a MURF-1 polypeptidecomprising, (i) transforming a host cell with an expression cassettecomprising the DNA segment of claim 1 and a promoter active in said hostcell and capable of directing the expression of said polypeptide, and,(ii) culturing the host cell under conditions suitable for theexpression of said polypeptide.